JP3600891B2 - Animal species discrimination method by SINE method - Google Patents

Description

に示す。
フタコブラクダは東京大学医学部の吉田穣博士より提供された筋肉組織からＤＮＡを抽出した。ブタについては当研究室に保管されていたＤＮＡをそのまま用いた。ペッカリーは農水省畜産試験場の安江博博士より提供されたＤＮＡを用いた。ジャワマメジカは米国サンディエゴ動物園から提供されたＤＮＡを用いた。アクシスジカ、アミメキリン、セーブルアンテロープ、ニホンカモシカ、マーコール、ムフロン、カバに関しては千葉市立動物公園の宗近功氏より提供されたＤＮＡを用いた。ヒツジは長野県上田食肉衛生検査場の向井康氏より提供された肝臓から抽出したものを使用した。ウシに関しては神奈川県食肉衛生試験場相模出張所より提供された腎臓からＤＮＡを抽出して用いた。
【００５０】
イッカク、オオギハクジラのサンプルについては、千葉県立中央博物館の宮正樹博士より筋肉と思われる組織片を入手しＤＮＡを抽出して用いた。オオギハクジラのサンプルに関してはアカボウクジラ科オオギハクジラ属であることは判明しているが種名は不明である。カワイルカ類を除くその他のサンプルは、水産庁遠洋水産研究所大型鯨類研究室の加藤秀弘博士より提供された各種組織片からＤＮＡを抽出して用いた。カワイルカ類のサンプルについては、アメリカ合衆国南西漁業科学センターのブラウネル博士（Ｄｒ． Ｒｏｂｅｒｔ Ｌ． Ｂｒｏｗｎｅｌｌ， Ｊｒ．： Ｃｈｉｅｆ Ｍａｒｉｎｅ Ｍａｍｍａｌ Ｄｉｖｉｓｉｏｎ， Ｓｏｕｔｈｅａｓｔ Ｆｉｓｈｅｒｉｅｓ Ｓｃｉｅｎｃｅ Ｃｅｎｔｅｒ， Ｐ．Ｏ． ＢＯＸ ２７１， Ｌａ Ｊｏｌｌａ， Ｃａｌｉｆｏｒｎｉａ ９２０３８）により日本国への入手手続きを行ってもらった。それに基づいて、アマゾンカワイルカの皮下組織片、ラプラタカワイルカの肝臓組織片についてはカリフォルニア州立大学バークレー校のＨｅａｌｙ Ｈ． Ｈａｍｉｌｔｏｎ 氏より提供を受けＤＮＡを抽出した。ガンジスカワイルカは乾燥骨サンプルを国立科学博物館の山田格氏より提供されＤＮＡを抽出した。ヨウスコウカワイルカに関しては中国科学院水生生物研究所の付属水族館に飼育されている個体から鮮血を採取しその場でヘパリン処理したものを研究所へ搬送し即座にＤＮＡを抽出した。抽出したＤＮＡは、日本への持ち込みが不可能なため現在は中国の研究所において保管されている。
【００５１】
ゲノム ＤＮＡ の抽出
解析に用いた動物種の組織からのゲノムＤＮＡ抽出は、簡便法（Ｒａｐｉｄ法；ＰｒｏｔｅｉｎａｓｅＫを用いる方法）で行った。抽出したゲノムＤＮＡはライブラリーの作製及びＰＣＲの鋳型として使用した。また、それらは４℃で保管している。−４０℃のフリザーに保存しておいた鯨、偶蹄類の組織（肝臓、筋肉）をメスで数ミリ角に切り出し、マイクロチューブに移してから組織の重量を測った後、そのマイクロチューブに１Ｘ ＴＮＥ ｂｕｆｆｅｒ （１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０），１００ｍＭ ＮａＣｌ， １ｍＭ ＥＤＴＡ （ｐＨ ８．０））を５００μｌ加えた。チューブ内の組織片をパスツールピペットを用いて細かく粉砕したらその、組織及びＴＮＥ ｂｕｆｆｅｒに１Ｘ Ｌｙｓｉｓ ｂｕｆｆｅｒ （２０ｍｇ／ｍｌ Ｐｒｏｔｅｉｎａｓｅ Ｋ，２％ ＳＤＳ， １０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）， １５０ｍＭ ＮａＣｌ， １０ｍＭ ＥＤＴＡ （ｐＨ８．０）） を加え、転倒撹拌した後、５５℃のウォーターバスを用いてインキュベートし組織片を完全に溶解させた。その溶液と等量のフェノールを加え、ベリーダンサー（２００〜３００ｒｐｍ）で１〜２時間撹拌した。遠心分離後 （室温、１２０００ｒｐｍ， ５ｍｉｎ）， 上清を別の２．０ｍｌ マイクロチューブに移した。同様にしてフェノール／クロロホルム抽出に続きクロロホルム抽出を繰り返し、最終的な上清に０．１倍量の３Ｍ 酢酸ナトリウム及び２倍量のエタノールを加えた。この際多くの場合ファイバーが確認され、その後７０％エタノールを用いてリンスした後適当量のＴＥ ｂｕｆｆｅｒを加えペレットを溶解させた。ヨウスコウカワイルカの血液サンプルからのＤＮＡの抽出は市販のカラム（ＱＩＡａｍｐ Ｔｉｓｓｕｅ／Ｂｌｏｏｄ Ｋｉｔ Ｃａｔ． Ｎｏ．２９３０６： キアゲン（株）社製）を用いて行った。
【００５２】
フランキング ＰＣＲ （主に相同遺伝子座の増幅に用いる）
フランキングＰＣＲとはＳＩＮＥ配列を挟む様にその両側（上流及び下流）の近傍領域にプライマーを設計し、ゲノムＤＮＡを鋳型としてＰＣＲを行う事を意味している。その際に用いるプライマーは長さにしておよそ１７〜３０ヌクレオチドでＴｍ（Ｍｅｌｔｉｎｇ Ｔｅｍｐｅｒａｔｕｒｅ）値はおおよそ５５℃周辺になるように設計した。その設計に関しては、マッキントッシュ版フリーソフトとしてインターネット上でダウンロードが可能なＣＰｒｉｍｅｒ （Ｖｅｒ．１．０８， Ｂｒｉｓｔｏｌ ａｎｄ Ａｎｄｅｒｓｏｎ（１９９５））を使用した。
反応組成：Ｔｅｍｐｌａｔｅ ＤＮＡ （１００ 〜 ５００ ｎｇ）； １０ Ｘ ＰＣＲ Ｂｕｆｆｅｒ （１００ ｍＭ Ｔｒｉｓ − ＨＣｌ （ｐＨ８．３）， ５００ ｍＭ ＫＣｌ， １５ ｍＭ ＭｇＣｌ_２ ５ μｌ； ｄＮＴＰ Ｍｉｘｔｕｒｅ （２．５ ｍＭ ｅａｃｈ） ４ μｌ； Ｆｏｒｗａｒｄ ａｎｄ Ｒｅｖｅｒｓｅ Ｐｒｉｍｅｒ （５ ｐｍｏｌ／μｌ） ２ μｌ ｅａｃｈ； ＴａＫａＲａ ＴａｑＴＭ （５ Ｕ／μｌ） ０．２５ μｌ； ａｄｄｓ ｄｄＨ_２Ｏ ｕｐ ｔｏ ｆｉｎａｌ ｖｏｌｕｍｅ ５０ μｌ。
反応条件： ９４ ℃（Ｐｒｅ−ｄｅｎａｔｕｒｅ） ２〜３ ｍｉｎ．； ａｎｄ ３０ ｃｙｃｌｅｓ ｏｆ ９４ ℃ （Ｄｅｎａｔｕｒｅ） ３０ ｓｅｃ．； ４５ ℃〜６０ ℃（Ａｎｎｅａｌｉｎｇ） １ ｍｉｎ； ７２ ℃ （Ｅｘｔｅｎｓｉｏｎ） ３０〜９０ ｓｅｃ．。この際Ａｎｎｅａｌｉｎｇの温度はプライマーのＴｍ値はもちろんのこと、その遺伝子座の増えやすさなどに応じて適宜変えていき最適な温度を探した。
【００５３】
コロニー ＰＣＲ
コロニーＰＣＲはサブクローニングを行う時、目的のＤＮＡ断片がインサートとしてあるクローンに含まれているか否かを選別するのに大変簡便な方法である。従来はプラスミドＤＮＡをＭｉｎｉｐｒｅｐによって調整した後に、制限酵素で消化してそのインサートの有無を確認していたが、この行程をＰＣＲによるチェックのみで済ませる事が可能となるので、時間と労力の大幅な短縮につながる。まずプレートに生えた大腸菌のコロニーを爪楊枝で軽くつつき０．５ ｍｌ ＰＣＲ 用マイクロチューブに擦り付け、それを鋳型にして通常のＰＣＲを２０ μｌの反応系にして行うだけである。この際用いるプライマーはベクターに特異的な配列に基づいて作製されている。ＰＣＲ反応における熱変性の際に大腸菌の細胞膜が破壊され鋳型となるベクターＤＮＡが溶液中に溶け出すことでこのＰＣＲが可能になる。後で述べるがこのＰＣＲによってインサートの有無を確認した後、このＰＣＲ産物を直接ＡＢＩシークエンサーを用いて配列決定の鋳型とすることが可能なので、インサートチェックの直後にそのインサートのシークエンスも可能なので時間的にもかなりの短縮になった。
【００５４】
塩基配列の決定
塩基配列の決定には以下に示すシークエンサー及びシークエンス反応キットを使用した。ＬＩ−ＣＯＲ ｄＮＡ Ｓｅｑｕｅｎｃｅｒ （Ｍｏｄｅｌ ４０００） を用いたシークエンス：ＳｅｑｕｉＴｈｅｒｍ ＥＸＣＥＬＬ（商標）ＩＩ Ｌｏｎｇ−ＲｅａｄＴＭ ＤＮＡ Ｓｅｑｕｅｎｃｅ Ｋｉｔ−ＬＣ （Ｃａｔ． Ｎｏ． ＳＥ７７０１ＬＣ， ＥＰＩＣＥＮＴＲＥ ＴＥＣＨＮＯＬＯＧＩＥＳ社製）；ＡＢＩ ＰＲＩＺＭ（商標） ３１０ Ｇｅｎｅｔｉｃ Ａｎａｌｙｓｅｒ を用いたシークエンス： ＢｉｇＤｙｅ Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ ＦＳ Ｒｅａｄｙ Ｒｅａｃｔｉｏｎ Ｋｉｔ （Ｐ／Ｎ ４３０３１５２， Ｐｅｒｋｉｎ Ｅｌｍｅｒ社製）。
サブクローニングしたプラスミドＤＮＡをシークエンス反応の鋳型に用いる場合は、 ＳＤＳ−Ａｌｋａｌｉｎｅ ａｎｄ Ｍｇ沈殿法を用いて調整したものを使用した。
スクリーニングによって単離したＳＩＮＥ配列を含んだ遺伝子座の塩基配列を決定する場合はベクター配列上のＭ４及びＲＶ、そしてＳＩＮＥのコンセンサス配列に基づいて作製した蛍光の付加しているオリゴヌクレオチドプライマー（アロカ（株）社製）を用いた。
ＰＣＲ産物をダイレクトシークエンスする場合は、ＰＣＲ産物にＳｈｒｉｍｐ Ａｌｋａｌｉｎｅ Ｐｈｏｓｐｈａｔａｓｅ （ＳＡＰ） ２Ｕ／μｌ ０．５μｌ； Ｅｘｏｎｕｃｌｅａｓｅ Ｉ １０Ｕ／μｌ ０．５ μｌ （共にアマシャムライフサイエンス社製）を直接加え、３７ ℃で３０ 分インキュベートしてオリゴヌクレオチドを分解した後、８５ ℃で１５ 分間インキュベートして酵素を失活させたものをシークエンス反応の鋳型として用いた。
【００５５】
ゲノム・ライブラリーの作製
スクロース勾配
ゲノムＤＮＡ約５０ μｇを適当な制限酵素（本研究においては主にＨｉｎｄ ＩＩＩを使用した）で完全消化し、フェノール／クロロホルム抽出、エタノール沈殿を行った後、２００ μ ｌ の ＴＥ ｂｕｆｆｅｒ に溶解した。１５ ｍｌ の超遠心分離用プラスチックチューブ （Ｃｅｎｔｒｉｆｕｇｅ Ｔｕｂｅｓ − ５０ Ｕｌｔｒａ − Ｃｌｅａｒ（商標）Ｔｕｂｅ １４ Ｘ ８９ ｍｍ， Ｏｒｄｅｒ Ｎｏ． ３４４０５９： ＢＥＣＫＭＡＮ 社製）に１０−４０ ％スクロース勾配を作製し、この上にＤＮＡ 溶液を加え、ローターに取り付け、超遠心分離機（Ｌ８−７０Ｍ， Ｓｅｒｉａｌ Ｎｏ． ７Ｃ８６９： ＢＥＣＫＭＡＮ 社製）で遠心分離（２５０００ ｒｐｍ．， １５ ℃， １５ 時間）した。遠心後、１．５ ｍｌ マイクロチューブに１０〜１５滴ずつ滴下して分画（約２５０〜４００ μｌ／フラクション）した。その分画を０．７〜１ ％アガロースゲル電気泳動し、約２〜４ ｋｂｐの断片を含むフラクションを挟むように４〜６本のフラクションチューブを選別しエタノール沈殿を行った後、適当量のＴＥ ｂｕｆｆｅｒに溶解した。再び０．７〜１ ％アガロース電気泳動を行い、ライブラリー作製に用いるフラクションを決定した。
【００５６】
プラスミド・ライブラリーの構築
今回の実験においてはｐＵＣ１８／ＨｉｎｄＩＩＩもしくはｐＵＣ１９／ＨｉｎｄＩＩＩを使用したプラスミド・ライブラリーのみを用い、ファージライブラリーは作製しなかった。それは鯨、偶蹄類ゲノム中にはＳＩＮＥのコピー数が十分量存在し、ファージライブラリーを使用しなくても十分量のポジティブクローンが得られる事が、当研究室での経験上わかっていたため、時間の節約を考えて比較的操作行程の少ないプラスミド・ライブラリーを活用した。プラスミド・ライブラリーの作製は以下の様にして行った。組成はＴａＫａＲａ Ｌｉｇａｔｉｏｎ Ｋｉｔ Ｖｅｒ．１ Ａ液， １２ μｌ； 同 Ｂ液， １．５ μｌ； Ｖｅｃｔｏｒ ＤＮＡ （ｐＵＣ１８ ｏｒ １９／ＨｉｎｄＩＩＩ〜１００ ｎｇ／μｌ）， ０．５ μｌ； Ｉｎｓｅｒｔ ＤＮＡ （Ｇｅｎｏｍｉｃ ＤＮＡ Ｓｕｃｒｏｓｅ Ｄｅｎｓｉｔｙ Ｇｒａｄｉｅｎｔ Ｆｒａｃｔｉｏｎ）， １．０ μｌ ： 以上の溶液を０．５ ｍｌチューブ中で混合した後、クールブロック上で１６ ℃で３０分以上放置した。このゲノム・ライブラリーは−２０ ℃で保存している。
【００５７】
ＳＩＮＥ 配列を含むクローン単離までの流れ
図１６にＳＩＮＥ法における実験の流れを示す。
スクリーニング
スクリーニングに使用するメンブレンの作製
０．５ ｍｌチューブに前述のゲノム・ライブラリー混合液を１ μｌ、大腸菌（ Ｅ．ｃｏｌｉ ＪＭ１０５株）のコンピテントセルを適当量加えて混合した後、氷上で３０分間静置した。４２〜４５ ℃で ４５ 秒間ヒートショックした後、あらかじめ ３７ ℃ でインキュベートしておいた Ｌ／ａｍｐ／Ｘ−ｇａｌ／ＩＰＴＧ プレートにプレーティングし、３７ ℃インキュベーターに一晩放置した。プレート１枚当たりのコロニー数が２００〜３００個になるように調整し、プレート１０〜２０枚分のライブラリーをまいた。このプレート上に生えたコロニーをナイロンメンブレン（Ｃｏｌｏｎｙ／Ｐｌａｑｕｅ Ｓｃｒｅｅｎ（商標）ＮＥＦ−９７８：ＮＥＮ Ｒｅｓｅａｒｃｈ Ｐｒｏｄｕｃｔｓ 社製）にトランスファーし、変性溶液（０．４ Ｍ ＮａＯＨ， ０．６ Ｍ ＮａＣｌ）， 中和溶液（１ Ｍ ＮａＣｌ， ０．５ Ｍ Ｔｒｉｓ−ＨＣｌ （ｐＨ７．０）） の順に３分間程度放置した。そのメンブレンは水分を切った後よく乾燥させておいた。コロニーをトランスファーした後のプレートは３７℃でインキュベートして再度コロニーが十分に生えた状態にしておき、後述のポジティブクローンのピックアップをしやすいようにした。
【００５８】
スクリーニングに使用するプローブの作製
スクリーニングにはオリゴヌクレオチドもしくはＰＣＲ産物を使用した。実際に使用したオリゴヌクレオチド配列を以下の表２：
【表２】

に示す。その作製方法はまず、オリゴヌクレオチドの場合：ｄｄＨ２Ｏ ２７〜３７μｌ； Ｏｌｉｇｏ Ｎｕｃｌｅｏｔｉｄｅ （５ ｐｍｏｌ／μｌ） １ μｌ； １０ Ｘ Ｔ４ Ｐｏｌｙｎｕｃｌｅｏｔｉｄｅ Ｋｉｎａｓｅ Ｂｕｆｆｅｒ ５ μｌ； Ｔ４ ｐｏｌｙｎｕｃｌｅｏｔｉｄｅ Ｋｉｎａｓｅ ２μｌ； ［γ−３２Ｐ］ＡＴＰ ５〜１５μｌを０．５ｍｌチューブに混合した後、３７℃でインキュベートした。プローブとして用いたオリゴヌクレオチドの配列を上記表２に示す。ＰＣＲ産物を用いる場合（Ｐｒｉｍｅｒ−Ｅｘｔｅｎｓｉｏｎ法）： Ｔｅｍｐｌａｔｅ ＤＮＡ， ５００ μｇ； Ｆｏｒｗａｒｄ ｐｒｉｍｅｒ （１２．５ ｎｍｏｌ／μｌ） ２ μｌ； Ｒｅｖｅｒｓｅ ｐｒｉｍｅｒ （１２．５ ｎｍｏｌ／μｌ） ２ μｌ ｄＤＴＰ Ｍｉｘｔｕｒｅ （ｄＡＴＰ， ｄＧＴＰ， ｄＴＴＰ） ２．５ μｌ； 以上の混合液を９５ ℃で５分間熱変性し、続いて５５ ℃で１分間アニールさせた後氷上に移した。さらに以下の試薬を順に混合した後６０ ℃で３０分間インキュベートした。１０ＸＢｃａＢＥＳＴ（商標）Ｂｕｆｆｅｒ ２．５ μｌ； ＢｃａＢＥＳＴ（商標）ＤＮＡ ｐｏｌｙｍｅｒａｓｅ ２ μｌ； ［α− ３２Ｐ］ ｄＣＴＰ。反応後のそれぞれのプローブはＮＩＣＫ Ｃｏｌｕｍｎ （Ｓｅｐｈａｄｅｘ Ｇ−５０ ＤＮＡ Ｇｒａｄｅ： アマシャムファルマシアバイオテク（株）社製）で溶出させて精製した。ＲＩのカウントは液体シンチレーションカウンターで測定した。（簡易的測定にガイガーカウンターも使用した。）
【００５９】
ハイブリダイゼーション
メンブレンをハイブリバックに入れ、そこにプレハイブリ溶液（６ Ｘ ＳＳＣ， １ ％ ＳＤＳ ） を適当量加えた。ハイブリバックをシーラーでパックし１時間以上インキュベートした。溶液を捨て、ハイブリ溶液（６ Ｘ ＳＳＣ， １ ％ ＳＤＳ， １ Ｘ Ｄｅｎｈａｒｔ’ｓ ｓｏｌｕｔｉｏｎ ， Ｃａｒｒｉｅｒ ＤＮＡ （Ｓｈａｒｅｄ Ｈｅｒｒｉｎｇ Ｓｐｅｒｍ ＤＮＡ ｓｏｌｕｔｉｏｎ）を加えた後、さらに予め調整したプローブを９５ ℃で３分間熱変性しておいたものを加え、またハイブリバックをシールした。その後４２ ℃のウォーターバスで一晩（〜１５時間程度）インキュベートした。それらのメンブレンを適当量のウォッシュ溶液（２Ｘ ＳＳＣ， １ ％ ＳＤＳ）で軽くすすぎカウントを測定し確認してから、さらに新たなウォッシュ溶液を用いてウォーターバスの温度を５５〜６０ ℃に設定してウォッシュを行った。
【００６０】
現像
ウォッシュ溶液を捨て新たなウォッシュ溶液で軽くすすいだ後、カウントを、ガイガーカウンターを用いて確認した。暗室内で台紙、メンブレン、Ｘ線フィルム、Ｉｎｔｅｎｓｉｆｉｅｒの順にカセットを入れ、これを−８０ ℃のフリザーに入れて感光させた。暗室内でカセットからＸ線フィルムを取り出し、現像液、停止液、定着液の順にＸ線フィルムを入れて現像した。
【００６１】
Ｐｏｓｉｔｉｖｅ Ｃｌｏｎｅ の単離
現像したＸ線フィルムとライブラリーをまいたプレートを重ね合わせる様にしてそのポジティブクローンの位置を確認した。そのポジティブクローンだと思われるコロニーについてはＭｉｎｉｐｒｅｐを行う前に、そのクローン中におけるＳＩＮＥ配列の有無の確認のためにベクターの配列ではなく、スクリーニングに用いたＳＩＮＥ配列のコンセンサス配列に基づいて作製したプライマーを用いてコロニーＰＣＲを行った。その後、ポジティブクローンのプラスミドを調製し、ＬＩ−ＣＯＲシークエンサーを用いてその近傍領域の配列決定を行った。その後の操作に関しては上述の通りであるので、以下、簡単に述べる。
【００６２】
まず、そのフランキング配列を基にしてプライマーを作製し、種々の生物のゲノムＤＮＡを鋳型にしてフランキングＰＣＲを行った。そして多くの場合、スクリーニングに用いた生物種の近縁種に関しては相同遺伝子座を簡単に増幅させることが可能であるが、それとは遠縁のグループもしくは分岐が古い時代に起こったと考えられる種の相同遺伝子座については、その近傍領域における塩基置換がより多く蓄積しているのでスクリーニングに用いた１種の配列に基づいたプライマーではアニールがうまくいかず、最初のフランキングＰＣＲでは増幅できない場合もしばしば見られた。その際には増幅した様々な種の相同遺伝子座の塩基配列を決定してそれらのコンセンサスをとりそれを考慮してまた新たなフランキングプライマーを作製し再度ＰＣＲを行った。
【００６３】
鯨・偶蹄目間の系統関係の推定の際にはアガロース・ゲル電気泳動の後、増幅されたバンドが相同遺伝子座なのか否かを確認するためにサザンハイブリダイゼーションを行った。その行程は以下に示す。アガロース・ゲル電気泳動後エチジウム・ブロマイド（ＥｔＢｒ）溶液で染色し、デジタルカメラによって泳動パターンを確認、撮影した。その後変性溶液（０．４ ＮａＯＨ， ０．６Ｍ ＮａＣｌ） に十分浸したメンブレン１枚、ろ紙２枚をこの順で、間に気泡が入らないようにゲルにのせてその上にＪＫワイパー、キムタオル（商標）、重しとして辞典などを載せ、一晩（〜１５時間）放置した。メンブレンを中和溶液（１ Ｍ ＮａＣｌ， ０．５ Ｍ Ｔｒｉｓ − ＨＣｌ （ｐＨ ７．０））に１５分程度放置して中和した後十分に乾燥させた。その後のハイブリダイゼーション、現像などの操作は前述のスクリーニングの時と同じなので省略する。また鯨目内の系統関係推定の際には殆どの遺伝子座についてそれらの塩基配列を決定したので基本的にサザンハイブリダイゼーションによる確認は行わなかった。
【００６４】
実施例１：鯨偶蹄目ゲノム内における ＣＨＲ−２ 各サブファミリーの分布
鯨の起源ついてはその内部系統について形態による分類と分子による分類で見解がわかれている。鯨目は現生の多くの種類を含む歯鯨亜目と髭鯨亜目、そしてこれらの祖先となったと考えられている原鯨亜目（ムカシクジラ亜目とも呼ばれている）の３つの亜目に分けられており（Ｆｏｒｄｙｃｅ ｅｔ ａｌ．， １９９４）、現生の鯨類の分類はその分類名からも明らかなように、歯を持つか否かによって区別されている。しかし、髭鯨類も発生のかなり最後の方の段階まで歯が確認できるし原鯨類の化石種の中には当然のことながら歯鯨と髭鯨の中間段階にあると思われるような形質を備えたもの（つまり歯を持った髭鯨）も存在しているので、実際の問題として歯の存在だけで歯鯨の単系統性を主張することはできない。それは系統的に考えた時に髭鯨の「髭」はおそらく共有派生形質であるが歯鯨の「歯」という形質は原始形質とみなされるためである。しかし歯鯨のみがエコロケーションをする能力を持ち、それに付随するメロン体の存在も歯鯨の単系統性を示唆する最も有名な形質のひとつである。
【００６５】
形態的分類が歯鯨、髭鯨類のそれぞれの単系統性を強く主張しているのに対して、現在までに行われてきた分子を用いた系統解析によればその殆どが歯鯨が単系統群を形成しないことを示唆している。その中でも大きな論争の火種となったのがＭｉｌｉｎｋｏｖｉｔｈ ａｎｄ Ｍｅｙｅｒ（１９９３）のミトコンドリア遺伝子配列の比較解析により提唱された系統樹でこの解析によれば歯鯨に含まれているマッコウクジラ上科のグループが他の歯鯨類よりむしろ髭鯨類に近縁である（つまり歯鯨は多系統である）という。この結果に続いて多くの分子統計学的研究がなされたが多くは歯鯨が多系統もしくは解決不可能という結果となっていた（ｅ． ｇ．， Ａｒｎａｓｏｎ ｅｔ ａｌ．， １９９４； Ａｄａｃｈｉ ｅｔ ａｌ．， １９９６； Ｓｍｉｔｈ ｅｔ ａｌ．， １９９６：図１７参照）。そこで、以下の問題を解決すべくＳＩＮＥ法を用いて種判別を行った：問題（１）歯鯨の単系統性の問題；（２）全てのカワイルカ類を含めたそれらの鯨目における系統関係；（３）鯨類それぞれの分岐年代。本願発明により、系統関係の決定のみならず現在まで絶対的な信頼性をもって進められてきた統計学的解析の問題点なども明らかにすることができた。
【００６６】
上述のように、本願発明者は、偶蹄目ゲノム中に存在するＣＨＲ−２には大きく分けてＦＬ（Ｆｕｌｌ Ｌｅｎｇｔｈ）， ＭＤ（Ｍｉｄｄｌｅ Ｄｅｌｅｔｉｏｎ ｔｙｐｅ）、ＤＴ（Ｄｅｌｅｔｉｏｎ Ｔｙｐｅ）、ＣＤ（Ｃｅｔａｃｅａ Ｄｅｌｅｔｉｏｎ ｔｙｐｅ）、 ＣＤＯ（Ｃｅｔａｃｅａ Ｄｅｌｅｔｉｏｎ ｔｙｐｅ Ｏｄｏｎｔｏｃｅｔｉ）のサブファミリーが存在することを発見した。それぞれのサブファミリーは進化の過程における増幅時期が異なるためそれらの分布は系統関係を反映すると考えられる。各サブファミリーのコンセンサス配列のアラインメントを図１８に示す。図１８から明らかなように各サブファミリーはそれらにＤｉａｇｎｏｓｔｉｃな塩基置換や欠失が認められる。
【００６７】
これらＳＩＮＥ各サブファミリーの鯨、偶蹄目における分布を確認するためにドットハイブリダイゼーションを行った。用いたプローブは、ＣＤとＣＤＯに関してはそれらの区別が可能な位置にオリゴプローブを設計し（アラインメントの太線）、ＦＬに関してはＰＣＲ産物を使用した。ドットハイブリダイゼーションの結果を図１２に示す。まずＦＬは全ての鯨目そして偶蹄目ではカバ、ウシ（反すう亜目を代表）のみに分布し、ブタやラクダ及びその他外群として加えた哺乳類ゲノム中には存在しないことが示唆された。次にＣＤは鯨目のみに特異的に分布し偶蹄目には分布してないと考えられ、鯨目の単系統性を強く支持している。さらにＣＤＯに関しては、歯鯨にのみ強くシグナルが確認されることからこのサブファミリーは歯鯨亜目特異的に爆発的な増幅をしたと考えられ、分子による解析から疑問視されている歯鯨の単系統性を支持するデータである。しかしドットハイブリダイゼーションによる解析においてシグナルが確認されないだけでは、単にその系統でコピー数が少なかっただけという可能性を否定できないので、その結果だけで系統関係を推定することはできない。そこで実際にそれらのＳＩＮＥが鯨目ゲノム中に挿入している様な遺伝子座を単離して系統関係の推定を行った。
【００６８】
実施例２−１： ＳＩＮＥ のゲノム中への挿入を指標とした鯨目内部系統の解析
鯨目内部系統に関する研究においては、鯨目に存在するＳＩＮＥの中から鯨目全体に特異的に分布しているＣＤ及び、歯鯨亜目に特異的に増幅していると考えられるＣＤＯに注目し、鯨目各種のゲノム・ライブラリーからこれらのサブファミリーに属するＳＩＮＥ配列を含む遺伝子座の単離を行った。上述のように効率的に系統解析を行うにはサブファミリーの分布とその増幅時期を対応させて予測ができる。マッコウクジラの系統解析に関しては、歯鯨の単系統性を示唆するような遺伝子座の探索を続けられてきたにもかかわらずそのような遺伝子座が単離できなかった理由として、主に歯鯨にのみ分布しているＣＤＯに注目して行っていたからではないかと考えた。そこでマッコウクジラのゲノム・ライブラリーのスクリーニングだけはＣＤＯより一つ前に増幅したと思われるＣＤの配列を用いて行った。以下に記載する各遺伝子座の名称と使用したゲノム・ライブラリーがどの鯨種由来かの対応関係をまとめておく：Ｍａｇｏ：マゴンドウ、Ｉｓｉ：イシイルカ、 Ａｍｚ：アマゾンカワイルカ、Ｔｕｔｉ：ツチクジラ、Ｓｐ 及びＳｐｅｒｍ：マッコウクジラ、Ｂａｎｄｏ：バンドイルカ、並びに Ｈｕｍｐ：ザトウクジラ。鯨目の系統関係を示唆する遺伝子座の単離に用いたＰＣＲプライマーを以下の表３：
【表３】

に示す。
【００６９】
実施例２−２：鯨目の単系統性を支持する遺伝子座
遺伝子座Ｂａｎｄｏ１をバンドウイルカ、遺伝子座Ｓｐ３１６をマッコウクジラのゲノム・ライブラリーから単離した。これらの遺伝子座の塩基配列決定、ＳＩＮＥ近傍領域におけるプライマー設計、鯨偶蹄目のゲノムＤＮＡを鋳型としたＰＣＲ反応、アガロース・ゲル電気泳動による分離を行った。その結果、図１９に示す通り遺伝子座Ｂａｎｄｏ１とＳｐ３１６では鯨目に共通にＳＩＮＥの挿入があったようなバンドパターンが確認できた。最も近縁な外群として用いたカバにはこの挿入はないので、鯨目が共通祖先であった時期にその挿入が起こったものと考えられる。これらの遺伝子座は鯨目の単系統性を強く支持している。遺伝子座Ｂａｎｄｏ１においてはまずＣＤが鯨目の共通祖先時に挿入し、その後南米のカワイルカ２種アマゾンカワイルカ、ラプラタカワイルカの共通祖先種においてＣＤＯの挿入があったものと考えられる。よってこの遺伝子座は鯨目の単系統性だけでなく南米カワイルカの単系統性も同時に示唆している。遺伝子座Ｓｐ３１６ではＣＨＲ−１ｔｙｐｅＩＩＩが鯨目の共通祖先で挿入ことを示している。図２０〜２２に遺伝子座Ｂａｎｄｏ１のアラインメントの結果を、そして図２３に遺伝子座Ｓｐ３１６のアラインメントの結果を示す。
【００７０】
実施例２−３：イルカ上科の単系統性を支持する遺伝子座
遺伝子座Ｍａｇｏ１９においてはイルカ上科４種（マイルカ科：コビレゴンドウ、バンドウイルカ；ネズミイルカ科：イシイルカ；イッカク科：イッカク）に特異的なＣＤＯの挿入が確認された（図２４参照）。この遺伝子座はイルカ上科の単系統性を強く支持している。図２５と２６に遺伝子座Ｍａｇｏ１９のアラインメントの結果を示す。
【００７１】
実施例２−４： Ｉｎｆｒａｏｒｄｅｒ： Ｄｅｌｐｈｉｎｉｄａ を支持する遺伝子座
遺伝子座Ｍａｇｏ２４， Ｍａｇｏ２６， Ｍａｇｏ３２， Ｉｓｉ１４， Ｉｓｉ３６， Ｉｓｉ３８ではＣＤＯの挿入がイルカ上科４種、南米カワイルカ２種アマゾンカワイルカ、ラプラタカワイルカ及びヨウスコウカワイルカにのみ確認された（図２７と２８参照）。よってこれらの遺伝子座はこのグループの単系統性を支持している。Ｍｕｉｚｏｎ（１９８８，１９９４）は形態的特徴からこれらのグループをＤｅｌｐｈｉｎｉｄａ下目（亜目よりも階級的には下）としてまとめているがこの考えにもよく一致している。またＡｒｎａｓｏｎ（１９９６）らのミトコンドリアＣｙｔｂ遺伝子の配列による解析でもこのことは強く支持されている。またカワイルカ類が多系統群でガンジスカワイルカが他のカワイルカ類より原始的であることを示唆している。図２９に遺伝子座Ｉｓｈｉ１４のアラインメントの結果を示す。図３０〜３１に遺伝子座Ｉｓｈｉ３６のアラインメントの結果を示す。図３２〜３３に遺伝子座Ｉｓｈｉ３８のアラインメントの結果を示す。図３４〜３５に遺伝子座Ｍａｇｏ２４のアラインメントの結果を示す。図３６〜３７に遺伝子座Ｍａｇｏ２６のアラインメントの結果を示す。図３８〜３９に遺伝子座Ｍａｇｏ３２のアラインメントの結果を示す。
【００７２】
実施例２−５：アカボウクジラ科の系統関係を示唆する遺伝子座
遺伝子座Ｍａｇｏ８， Ｍａｇｏ１３においてはＤｅｌｐｈｉｎｉｄａとアカボウクジラ科に属する２種ツチクジラ、オオギハクジラに特異的なＣＤＯの挿入が確認された（図４０参照）。このことから現生種ではアカボウクジラ科がＤｅｌｐｈｉｎｉｄａに最も近縁な分類群であるといえる。またガンジスカワイルカ科はアカボウクジラ科よりさらに原始的なグループであることがわかる。図４１に遺伝子座Ｍａｇｏ８のアラインメントの結果を示す。図４２に遺伝子座Ｍａｇｏ１３のアラインメントの結果を示す。
【００７３】
実施例２−６：ガンジスカワイルカ科の系統関係を示唆する遺伝子座
遺伝子座Ｍａｇｏ２１， Ｍａｇｏ２２においてはＤｅｌｐｈｉｎｉｄａ、アカボウクジラ科及びガンジスカワイルカに特異的なＣＤＯの挿入が確認された（図４３参照）。よってガンジスカワイルカ科（インダスカワイルカはサンプルがないため解析には加えていないがおそらくガンジスカワイルカと単系統群を形成する。）はマッコウクジラを除く歯鯨の中では最も原始的なグループであることが明らかとなった。図４４に遺伝子座Ｍａｇｏ２１のアラインメントの結果を示す。図４５〜４７に遺伝子座Ｍａｇｏ２２のアラインメントの結果を示す。
【００７４】
実施例２−７：歯鯨の単系統性を支持する遺伝子座
遺伝子座Ｓｐｅｒｍ８， Ｓｐｅｒｍ２８， Ｓｐｅｒｍ４７においてはマッコウクジラも含めた歯鯨亜目全ての種で共通にＣＤの挿入が確認された（図４８参照）。これらの遺伝子座の解析結果は分子統計学的研究から疑問視され続けてきた歯鯨の単系統性が強く支持された。図４９〜５０に遺伝子座Ｓｐｅｒｍ８のアラインメントの結果を示す。図５１に遺伝子座Ｓｐｅｒｍ２８のアラインメントの結果を示す。図５２に遺伝子座Ｓｐｅｒｍ４７のアラインメントの結果を示す。
【００７５】
実施例２−８：南米カワイルカの単系統性を支持する遺伝子座
遺伝子座Ａｍｚ１３， Ｂａｎｄｏ１においては南米カワイルカ２種アマゾンカワイルカ、ラプラタカワイルカにおいて共通にＣＤＯの挿入が見られた（図５３参照）。南米カワイルカ２種及びヨウスコウカワイルカの３者の系統関係に関してはいくつかの仮説がありヨウスコウカワイルカが南米カワイルカ類のいずれかと単系統群を形成する説もあったが（Ｋａｓｕｙａ， １９７３； Ｂａｒｎｅｓ ｅｔ ａｌ．， １９８５）これら２つの遺伝子座によって南米カワイルカ類が単系統であることが強く支持された。今までに提唱されてきたいくつかの仮説の中では生物地理学的にも最も理想的な系統関係であると考えられる。図５４に遺伝子座Ａｍｚ１３のアラインメントの結果を示す。
【００７６】
実施例２−９：アマゾンカワイルカに特異的な遺伝子座 Ａｍｚ１１
図５５にアマゾンカワイルカに特異的にＣＤＯの挿入があったことを示す遺伝子座Ａｍｚ１１のバンドパターンを示す。図５６に遺伝子座Ａｍｚ１１のアラインメントの結果を示す。
【００７７】
実施例２−１０：アカボウクジラ科に特異的な遺伝子座 Ｔｕｔｉ２４， Ｔｕｔｉ３５
図５７にアカボウクジラ科に特異的にＣＤＯの挿入があったことを示す遺伝子座Ｔｕｔｉ２４， Ｔｕｔｉ３５のバンドパターンを示す。図５８に遺伝子座Ｔｕｔｉ２４のアラインメントの結果を示す。図５９〜６０に遺伝子座Ｔｕｔｉ３５のアラインメントの結果を示す。
【００７８】
実施例２−１１：マッコウクジラ上科３種に特異的な遺伝子座 Ｓｐ９ 、及びマッコウクジラ科に特異的な Ｓｐ２
図６１にマッコウクジラ上科３種にＣＤＯの挿入があったことを示す遺伝子座Ｓｐ９、及びマッコウクジラ科に特異的なＳｐ２のバンドパターンを示す。図６２〜６３に遺伝子座Ｓｐ２のアラインメントの結果を示す。図６４に遺伝子座Ｓｐ９のアラインメントの結果を示す。
【００７９】
実施例２−１２：ナガスクジラ科（又は髭鯨）に特異的な遺伝子座 Ｈｕｍｐ２０， Ｈｕｍｐ２０３
図６５にナガスクジラ科（又は髭鯨）に特異的なＣＤの挿入があったことを示す遺伝子座Ｈｕｍｐ２０， Ｈｕｍｐ２０３のバンドパターンを示す。図６６〜６８に遺伝子座Ｈｕｍｐ２０のアラインメントの結果を示す。図６９に遺伝子座Ｈｕｍｐ２０３のアラインメントの結果を示す。
【００８０】
実施例３：鯨目内部の系統樹の作成
以上の結果から得られたＳＩＮＥの挿入パターンをマトリックスにまとめた（図７０参照）。これに基づき推定される鯨目内部の系統発生樹を図７１に示す。
それぞれの遺伝子座の解析の際に得られたＰＣＲ産物の塩基配列は可能な限りすべて決定し、アラインメントした。その結果得られたバンドパターンが２次的な挿入や欠失などによってもたらされたものでないことを確認した。またこれらの配列は鯨目の核遺伝子配列情報として今後の研究に重要であると考えている。
【００８１】
結果
上記実施例によって明らかになった現生歯鯨類の系統関係を以下にまとめる。１．歯鯨類は単系統群を形成する。マッコウクジラ上科は現生歯鯨の中で最初に分岐したグループである。
２．ガンジスカワイルカ（おそらくインダスカワイルカと単系統）がマッコウクジラ類の次に分岐した。他のカワイルカ類とは別の系統群から派生したものである。
３．次にアカボウクジラ科のグループが分岐する。またアカボウクジラ科のＢｅ ｒａｄｉｕｓ属及びＭｅｓｏｐｌｏｄｏｎ属は単系統である。
４．南米のカワイルカ２種アマゾンカワイルカ、ラプラタカワイルカが単系統である。
５．イルカ上科に属するマイルカ科、ネズミイルカ科及びイッカク科は単系統群を形成する。
【００８２】
以上の系統関係は、系統発生学的に重要な意味をもつだけではなく、数種の動物から成る検体が提供された場合、ＳＩＮＥが挿入された適当な遺伝子座を選択することにより、塩基配列分析を省略してその検体がどの種であるか判別することができる本願発明に係るＳＩＮＥ法を用いた種判別方法の信頼性の基礎をなす。鯨目以外の動物についてもＳＩＮＥ法により、さらに系統発生樹を確立することによって、それらの目内においても鯨目内におけるものと同様な種判別方法を確立することができる。なぜなら、分子統計学的手法を用いた系統解析には以下に述べるように限界があるからである。
過去にマッコウクジラの系統関係に関して行われた分子統計学的な研究で歯鯨の単系統性を示唆するような結果は殆ど得られていなかった。これらの解析が主にミトコンドリア遺伝子の配列を用いていたことから当初はミトコンドリアに特異的な現象なのかとも考えられたが、ミオグロビンのアミノ酸配列の解析（Ｃｚｅｌｕｓｎｉａｋ ｅｔ ａｌ．， １９９０）でも同じ結果であったし、核遺伝子ＩＲＢＰの解析 （Ｓｍｉｔｈ ｅｔ ａｌ．， １９９６） でも歯鯨の多系統性（マッコウクジラがよりヒゲクジラに近縁である）の方が統計的に有利である結果を示していることから、分子配列を統計学的に解析するとどうしても歯鯨が多系統であるような結果が得られるようである（図７２参照）。しかし上記の結果は明らかに歯鯨が単系統群を形成する事を示唆しているし、また形態学的な特徴から考えても歯鯨の単系統性はまず間違いないであろう。
【００８３】
では何故鯨類を分子統計学的に解析するとマッコウクジラがヒゲクジラにより近縁であるような結果が出てしまうのだろうか。しばしば問題視されるのがアウトグループの選択である。つまり分子の情報からわかるのは各グループの分子的な距離だけでありそれらのグループ（この場合はイルカ、マッコウクジラ、ヒゲクジラ）が分岐する以前に分岐した外群を用いて系統樹の根（Ｒｏｏｔ）の位置を確定する必要がある。しかしその位置は外群の種類と数の違いによって異なる場合があり、初期のＡｒｎａｓｏｎ（１９９４）の解析のように外群をウシ一種しか用いない様な解析はかなり信頼度は低いと考えられる。実際にＣｙｔｂ遺伝子に関してはＡｄａｃｈｉとＨａｓｅｇａｗａ（１９９５）の研究によればアウトグループを変えることでその結果が大きく変わることがわかっている。しかしその後の多数の偶蹄目及びその他有蹄類を外群に用いて行った解析（Ａｒｎａｓｏｎ ｅｔ ａｌ．， １９９６）でも、歯鯨の単系統性を示唆することができなかったことから単にアウトグループの選択が問題であったとは考えにくい。次に鯨類それぞれの分類群の間で分子進化速度が異なる可能性が考えられる。Ｍａｒｔｉｎ（１９９３）らの研究では塩基置換速度に影響を及ぼすと考えられる代謝の影響などと生物の体のサイズの相関関係を示し、一般に体のサイズが大きいほど塩基置換速度が遅くなる傾向があることを示唆した。つまりイルカ類などの小型の歯鯨類がマッコウクジラやナガスクジラ類の様な大型の髭鯨類に比べて分子進化速度が大きいために統計的な誤差をもたらしたとも考えられる。しかし最終的にＨａｓｅｇａｗａ（１９９７）らが解析に用いた最尤法（Ｍａｘｉｍｕｍ ｌｉｋｅｌｉｈｏｏｄ ａｎａｌｙｓｉｓ）は最大節約法（Ｍａｘｉｍｕｍ ｐａｒｓｉｍｏｎｙ ａｎａｌｙｓｉｓ）とは異なり、各分類群間で分子進化速度が違うような場合においても結果的に間違えた系統樹をつくることは少ないのが特徴であるにもかかわらず結果は変わらなかった。
【００８４】
また種の分岐が短時間の間に起こった可能性も十分に考えられる。例えば歯鯨類ニ髭鯨類の分岐の後、極めて短時間で歯鯨類からマッコウクジラ類が分岐してしまったような場合は歯鯨の間で共通な塩基置換が蓄積するのに十分な時間がなく、しかもマッコウクジラ類がその後の現在に至るまでの長い時間を独自の歴史を経てしまえばその間に蓄積した塩基置換が、歯鯨が共通祖先の時期に蓄積したであろうわずかな共通塩基置換をうやむやにしてしまうことが考えられる。実際に今から３５００万年程前に起きた、オーストラリア大陸の北偏がもたらした南極海流の成立で鯨類の適応放散による種分化はかなりのスピードで起こったことが予想される（Ｆｏｒｄｙｃｅ， １９９２）。このような場合はどのような解析方法を用いても分子統計学的な解析による系統推定は不可能であると考えられる。また、もし様々な統計学的な仮定を前提にしてより確からしい系統推定ができたとしてもかなり人為的な系統樹になってしまうのではないだろうか。これでは形態的研究よりも勝っている「客観性」という観念からはずれてしまうように思われる。
【００８５】
【発明の効果】
本発明に係る動物種の判別方法の有利な効果の１つは、配列決定工程を省略できることである。一般に、シークエンス装置は最低でも１５００万円であり、そして消耗品については、１回のシークエンスにかかる費用はおよそ１２００円である。したがって、多数の検体を分析する場合、かなりの費用がかかる。さらに、検査会社により商業的に分析する場合、現状、１検体当たり１万円以上請求される。時間的にも、配列決定工程には１検体当たり少なくとも１時間３０分程かかるので、これが省略できるＳＩＮＥ法を用いた種判別方法は分析効率が高い。最も重要なことは、ＤＮＡ塩基配列の解析は統計的な操作を伴うということである。上述のように、このうような統計学的な手法はその解析方法によっても結果がことなるという問題が生じている。これに対し、ＳＩＮＥ法は、ＳＩＮＥの挿入という不可逆現象を指標として種判別を行うので、その結果は明瞭であり、特別な統計学的処理は必要とされない。結論として、本願発明に係るＳＩＮＥ法を用いた種判別方法は、従来技術の塩基配列の比較に基づく種判別方法よりも、金銭的に、時間的にさらにはその信頼性に関しても優れているといえる。
【００８６】
参考文献一覧
１．Ａｌｔｓｃｈｕｌ ＳＦ， Ｇｉｓｈ Ｗ， Ｍｉｌｌｅｒ Ｗ， Ｍｙｅｒｓ ＥＷ， Ｌｉｐｍａｎ ＤＪ． Ｂａｓｉｃ ｌｏｃａｌ ａｌｉｇｎｍｅｎｔ ｓｅａｒｃｈ ｔｏｏｌ． Ｊ． Ｍｏｌ． Ｂｉｏｌ． １９９０； ２１５：４０３−４１０
２．Ｂａｔｚｅｒ， Ｍ． Ａ． ｅｔ ａｌ． Ａｆｒｉｃａｎ ｏｒｉｇｉｎ ｏｆ ｈｕｍａｎ−ｓｐｅｃｉｆｉｃ ｐｏｌｙｍｏｒｐｈｉｃ Ａｌｕ ｉｎｓｅｒｔｉｏｎｓ． Ｐｒｏｃ． Ｎａｔ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ １９９４； ９１： １２２８８−１２２９２
３．Ｂｏｒｏｄｕｌｉｎａ ＯＲ， Ｋｒａｍｅｒｏｖ ＤＡ． Ｗｉｄｅ ｄｉｓｔｒｉｂｕｔｉｏｎ ｏｆ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｅｌｅｍｅｎｔｓ ａｍｏｎｇ ｅｕｋａｒｙｏｔｉｃ ｇｅｎｏｍｅｓ． ＦＥＢＳ Ｌｅｔｔ． １９９９； ４５７：４０９−１３
４．Ｂｒｉｔｔｅｎ ＲＪ， Ｂａｒｏｎ ＷＦ， Ｓｔｏｕｔ ＤＢ， Ｄａｖｉｄｓｏｎ Ｅｌｉ． Ｓｏｕｒｃｅｓ ａｎｄ ｅｖｏｌｕｔｉｏｎ ｏｆ ｈｕｍａｎ Ａｌｕ ｒｅｐｅａｔｅｄ ｓｅｑｕｅｎｃｅｓ． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ １９８８；８５：４７７０−４７７４
５．Ｂｒｏｓｉｕｓ Ｊ． Ｒｅｔｒｏｐｏｓｏｎｓ −− ｓｅｅｄｓ ｏｆ ｅｖｏｌｕｔｉｏｎ． Ｓｃｉｅｎｃｅ １９９１； ２５１： ７５３
６．Ｂｕｃｃｉ Ｓ， Ｒａｇｇｈｉａｎｔｉ Ｍ， Ｍａｎｃｉｎｏ Ｇ， Ｐｅｔｒｏｎｉ Ｇ， Ｇｕｅｒｒｉｎｉ Ｆ， Ｇｉａｍｐａｏｌｉ Ｓ． Ｒａｎａ／Ｐｏｌ ＩＩＩ： ａ ｆａｍｉｌｙ ｏｆ ＳＩＮＥ−ｌｉｋｅ ｓｅｑｕｅｎｃｅｓ ｉｎ ｔｈｅ ｇｅｎｏｍｅｓ ｏｆ ｗｅｓｔｅｒｎ Ｐａｌｅａｒｃｔｉｃ ｗａｔｅｒ ｆｒｏｇｓ． Ｇｅｎｏｉｎｅ １９９９； ４２：５０４−５１１
７．Ｃａｏ Ｙ， Ｆｕｊｉｗａｒａ Ｍ， Ｎｉｋａｉｄｏ Ｍ， Ｏｋａｄａ Ｎ， Ｈａｓｅｇａｗａ Ｍ． Ｉｎｔｅｒｏｒｄｉｎａｌ ｒｅｌａｔｉｏｎｓｈｉｐｓ ａｎｄ ｔｉｍｅｓｃａｌｅ ｏｆ ｅｕｔｈｅｒｉａｎ ｅｖｏｌｕｔｉｏｎ ａｓ ｉｎｆｅｒｒｅｄ ｆｒｏｍ ｍｉｔｏｃｈｏｎｄｒｉａｌ ｇｅｎｏｍｅ ｄａｔａ． ２０００ （ｉｎ ｐｒｅｓｓ）
８．Ｃｌａｒｋ ＪＢ， Ｋｉｄｗｅｌｌ Ｍ． Ａ ｐｈｙｌｏｇｅｎｅｔｉｃ ｐｅｒｓｐｅｃｔｉｖｅ ｏｎ Ｐ ｔｒａｎｓｐｏｓａｂｌｅ ｅｌｅｍｅｎｔ ｅｖｏｌｕｔｉｏｎ ｉｎ Ｄｒｏｓｏｐｈｉｌａ． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ １１９７； ９４：１１４２８−１１４３３
９．Ｃｏｌｔｍａｎ ＤＷ， Ｗｒｉｇｈｔ ＪＭ． Ｃａｎ ＳＩＮＥｓ： ａ ｆａｍｉｌｙ ｏｆ ｔＲＮＡ−ｄｅｒｉｖｅｄ ｒｅｔｒｏｐｏｓｏｎｓ ｓｐｅｃｉｆｉｃ ｔｏ ｔｈｅ ｓｕｐｅｒｆａｍｉｌｙ Ｃａｎｏｉｄｅａ． Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． １９９４；２２：２７２６−２７３０
１０．Ｄｅｉｎｉｎｇｅｒ ＰＬ， Ｂａｔｚｅｒ ＭＡ． Ｅｖｏｌｕｔｉｏｎ ｏｆ ｒｅｔｒｏｐｏｓｏｎｓ． Ｅｖｏｌ． Ｂｉｏｌ． １９９３；２７：１５７−１９６
１１．Ｄｅｒａｇｏｎ ＪＭ， Ｌａｎｄｒｙ ＢＳ， Ｐｅｌｉｓｓｉｅｒ Ｔ， Ｔｕｔｏｉｓ ５， Ｔｏｕｒｍｅｎｔｅ ５， Ｐｉｃａｒｄ Ｇ． Ａｎ ａｎａｌｙｓｉｓ ｏｆ ｒｅｔｒｏｐｏｓｉｔｉｏｎ ｉｎ ｐｌａｎｔｓ ｂａｓｅｄ ｏｎ ａ ｆａｍｉｌｙ ｏｆ ＳＩＮＥｓ ｆｒｏｍ Ｂｒａｓｓｉｃａ ｎａｐｕｓ． Ｊ． Ｍｏｌ． Ｅｖｏｌ． １９９４； ３９：３７８−３８６
１２．Ｅｉｃｋｂｕｓｈ ＴＨ． Ｏｒｉｇｉｎ ａｎｄ ｅｖｏｌｕｔｉｏｎａｒｙ ｒｅｌａｔｉｏｎｓｈｉｐｓ ｏｆ ｒｅｔｒｏｅｌｅｍｅｎｔｓ． Ｉｎ Ｍｏｒｓｅ ＳＳ． ｅｄ； Ｔｈｅ Ｅｖｏｌｕｔｉｏｎａｒｙ Ｂｉｏｌｏｇｙ ｏｆ Ｖｉｒｕｓｅｓ． Ｎｅｗ Ｙｏｒｋ： Ｒａｖｅｎ Ｐｒｅｓｓ． １９９４． Ｐｐ．１２１−１５７
１３．Ｇａｌｌａｇｈｅｒ ＰＣ， Ｌｅａｒ ＴＬ， Ｃｏｏｇｌｅ ＬＤ， Ｂａｉｌｅｙ Ｅ． Ｔｗｏ ＳＩＮＥ ｆａｍｉｌｉｅｓ ａｓｓｏｃｉａｔｅｄ ｗｉｔｈ ｅｑｕｉｎｅ ｍｉｃｒｏｓａｔｅｌｌｉｔｅ ｌｏｃｉ． Ｍａｍｍ Ｇｅｎｏｍｅ． １９９９； １０： １４０−１４４
１４．Ｇａｌｌｉ Ｇ， Ｈｏｆｓｔｅｔｔｅｒ Ｈ， Ｂｉｒｎｓｔｉｅｌ ＭＬ． Ｔｗｏ ｃｏｎｓｅｒｖｅｄ ｓｅｑｕｅｎｃｅ ｂｌｏｃｋｓ ｗｉｔｈｉｎ ｅｕｋａｒｙｏｔｉｃ ｔＲＮＡ ｇｅｎｅｓ ａｒｅ ｍａｊｏｒ ｐｒｏｍｏｔｅｒ ｅｌｅｍｅｎｔｓ． Ｎａｔｕｒｅ １９８１；２９４：６２６−６３１
１５．Ｈａｍａｄａ Ｍ， Ｔａｋａｓａｋｉ Ｎ， Ｒｅｉｓｔ ＪＤ， Ｄｅ Ｃｉｃｃｏ， Ｇｏｔｏ Ａ， Ｏｋａｄａ Ｎ． Ｄｅｔｅｃｔｉｏｎ ｏｆ ｔｈｅ ｏｎｇｏｉｎｇ ｓｏｒｔｉｎｇ ｏｆ ａｎｃｅｓｔｒａｌｌｙ ｐｏｌｙｍｏｒｐｈｉｃ ＳＩＮＥｓ ｔｏｗａｒｄ ｆｉｘａｔｉｏｎ ｏｒ ｌｏｓｓ ｉｎ ｐｏｐｕｌａｔｉｏｎｓ ｏｆ ｔｗｏ ｓｐｅｃｉｅｓ ｏｆ ｃｈａｒｒ ｄｕｒｉｎｇ ｓｐｅｃｉａｔｉｏｎ． Ｇｅｎｅｔｉｃｓ １９９８；１５０： ３０１−３１１
１６．Ｈａｒｔｌ ＤＬ， Ｌｏｈｅ ＡＲ， Ｌｏｚｏｖｓｋｙａ ＥＲ． Ｍｏｄｅｒｎ ｔｈｏｕｇｈｔｓ ｏｎ ａｎ ａｎｃｙｅｎｔ ｍａｒｉｎｅｒｅ： ｆｕｎｃｔｉｏｎ， ｅｖｏｌｕｔｉｏｎ， ｒｅｇｕｌａｔｉｏｎ． Ａｎｎｕ． Ｒｅｖ． Ｇｅｎｅｔ． １９９７； ３１：３３７−３５８．
１７．Ｈｅｎｄｙ ＭＤ， Ｐｅｎｎｙ Ｄ． Ｓｙｓｔ． Ｚｏｏｌ． １９８９；３８：２９７−０００
１８．Ｈｅｎｎｉｇ Ｗ． Ｐｈｙｌｏｇｅｎｅｔｉｃ Ｓｙｓｔｅｍａｔｉｃｓ． Ｕｒｂａｎａ−Ｃｈａｍｐａｇｎｅ： Ｕｎｉｖｅｒｓｉｔｙ ｏｆ Ｉｌｌｉｎｏｉｓ Ｐｒｅｓｓ． １９６６．
１９．Ｈｉｌｌｉｓ ＤＭ． ＳＩＮＥｓ ｏｆ ｔｈｅ ｐｅｒｆｅｃｔ ｃｈａｒａｃｔｅｒ． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ．， ＵＳＡ １９９９；９６： ９９７９−９９８１
２０．Ｉｚｓｖａｋ Ｚ， Ｉｖｉｃｓ Ｚ， Ｇａｒｃｉａ−Ｅｓｔｅｆａｎｉａ Ｄ， Ｆａｈｒｅｎｋｒｕｇ ＳＣ， Ｈａｃｋｅｔｔ ＰＢ． ＤＡＮＡ ｅｌｅｍｅｎｔｓ： ａ ｆａｍｉｌｙ ｏｆ ｃｏｍｐｏｓｉｔｅ， ｔＲＮＡ−ｄｅｒｉｖｅｄ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ＤＮＡ ｅｌｅｍｅｎｔｓ ａｓｓｏｃｉａｔｅｄ ｗｉｔｈ ｍｕｔａｔｉｏｎａｌ ａｃｔｉｖｉｔｉｅｓ ｉｎ ｚｅｂｒａｆｉｓｈ （Ｄａｎｉｏ ｒｅｒｉｏ）． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ １９９６；９３：１０７７− １０８１
２１．Ｊｕｒｋａ Ｊ， Ｚｅｉｔｋｉｅｗｉｃｚ Ｅ， Ｌａｂｕｄａ Ｄ． Ｕｂｉｑｕｉｔｏｕｓ ｍａｍｍａｌｉａｎ−ｗｉｄｅ ｉｎｔｅｒｓｐｅｒｓｅｄ ｒｅｐｅａｔｓ （ＭＩＲ５） ａｒｅ ｍｏｌｅｃｕｌａｒ ｆｏｓｓｉｌｓ ｆｒｏｍ ｔｈｅ Ｍｅｓｏｚｏｎｉｃ ｅｒａ． Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． １９９５；２３：１７０−１７５
２２．Ｋａｓｓ ＤＨ， Ｒａｙｎｏｒ ＭＥ， Ｗｉｌｌｉａｍｓ ＴＭ． Ｅｖｏｌｕｔｉｏｎａｒｙ ｈｉｓｔｏｒｙ ｏｆ Ｂ１ ｒｅｔｒｏｐｏｓｏｎｓ ｉｎ ｔｈｅ ｇｅｎｕｓ Ｍｕｓ． Ｊ Ｍｏｌ Ｅｖｏｌ． ２０００；５１：２５６−６４
２３．Ｋａｚａｚｉａｎ ＨＨ Ｊｒ， Ｍｏｒａｎ ＪＶ． Ｔｈｅ ｉｍｐａｃｔ ｏｆ Ｌｉ ｒｅｔｒｏｔｒａｎｓｐｏｓｏｎｓ ｏｎ ｔｈｅ ｈｕｍａｎ ｇｅｎｏｍｅ． Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ １９９８；１９：１９−２４
２４．Ｋｉｄｏ Ｙ， Ｈｉｍｂｅｒｇ Ｍ， Ｔａｋａｓａｋｉ Ｎ， Ｏｋａｄａ Ｎ． Ａｍｐｌｉｆｉｃａｔｉｏｎ ｏｆ ｄｉｓｔｉｎｃｔ ｓｕｂｆａｍｉｌｉｅｓ ｏｆ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｅｌｅｍｅｎｔｓ （ＳＩＮＥｓ） ｄｕｒｉｎｇ ｅｖｏｌｕｔｉｏｎ ｏｆ ｔｈｅ Ｓａｌｍｏｎｉｄａｅ． Ｊ． Ｍｏｌ． Ｂｉｏｌ． １９９４；２４１： ６３３−６４４
２５．Ｋｉｄｏ Ｙ， Ｓａｉｔｏｈ Ｍ， Ｍｕｒａｔａ ５， Ｏｋａｄａ Ｎ． Ｅｖｏｌｕｔｉｏｎ ｏｆ ｔｈｅ ａｃｔｉｖｅ ｓｅｑｕｅｎｃｅｓ ｏｆ ｔｈｅ ＨｐａＩ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｅｌｅｍｅｎｔｓ． Ｊ． Ｍｏｌ． Ｅｖｏｌ． １９９５；４１：９８６−９９５
２６．Ｋｉｍ Ｊ， Ｍａｒｔｉｇｎｅｔｔｉ ＪＡ， Ｓｈｅｎ ＭＲ， Ｂｒｏｓｉｕｓ Ｊ， Ｄｅｉｎｉｎｇｅｒ Ｐ． Ｒｏｄｅｎｔ ＢＣ１ ＲＮＡ ｇｅｎｅ ａｓ ａ ｍａｓｔｅｒ ｇｅｎｅ ｆｏｒ ＩＤ ｅｌｅｍｅｎｔ ａｍｐｌｉｆｉｃａｔｉｏｎ． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ．， ＵＳＡ １９９４；９１： ３６０７−３６１１
２７．Ｋｒａｍｅｒｏｖ Ｄ， Ｖａｓｓｅｔｚｋｙ Ｎ， Ｓｅｒｄｏｂｏｖａ Ｉ． Ｔｈｅ ｅｖｏｌｕｔｉｏｎａｒｙ ｐｏｓｉｔｉｏｎ ｏｆ ｄｏｒｍｉｃｅ （Ｇｌｉｒｉｄａｅ） ｉｎ Ｒｏｄｅｎｔｉａ ｄｅｔｅｒｍｉｎｅｄ ｂｙ ａ ｎｏｖｅｌ ｓｈｏｒｔ ｒｅｔｒｏｐｏｓｏｎ． Ｍｏｌ． Ｂｉｏｌ． Ｅｖｏｌ． １９９９；１６：７１５−７１７
２８．Ｌｅｅｆｌａｎｇ ＥＰ， Ｌｉｕ Ｗ−Ｍ， Ｈａｓｈｉｍｏｔｏ Ｃ， Ｃｈｏｕｄａｒｙ ＰＶ， Ｓｃｈｍｉｄ ＣＷ． Ｐｈｙｌｏｇｅｎｅｔｉｃ ｅｖｉｄｅｎｃｅ ｆｏｒ ｍｕｌｔｉｐｌｅ Ａｌｕ ｓｏｕｒｃｅ ｇｅｎｅｓ． Ｊ． Ｍｏｌ． Ｅｖｏｌ． １９９２；３５： ７−１６
２９．Ｌｕａｎ ＤＤ， Ｋｏｒｍａｎ ＭＨ， Ｊａｋｕｂｃｚａｋ ＪＬ， Ｅｉｃｋｂｕｓｈ ＴＨ． Ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ ｏｆ Ｒ２Ｂｍ ＲＮＡ ｉｓ ｐｒｉｍｅｄ ｂｙ ａ ｎｉｃｋ ａｔ ｔｈｅ ｃｈｒｏｍｏｓｏｍａｌ ｔａｒｇｅｔ ｓｉｔｅ： ａ ｍｅｃｈａｎｉｓｍ ｆｏｒ ｎｏｎ−ＲＴＬ ｒｅｔｒｏｔｒａｎｓｐｏｓｉｔｉｏｎ． Ｃｅｌｌ １９９３ ；７２： ５９５−６０５
３０．Ｌｕｍ ＪＫ， Ｎｉｋａｉｄｏ Ｍ， Ｓｈｉｍａｍｕｒａ Ｍ， Ｓｈｉｍｏｄａｉｒａ Ｈ， Ｓｈｅｄｌｏｃｋ ＡＭ， Ｏｋａｄａ Ｎ， Ｈａｓｅｇａｗａ Ｍ． Ｃｏｎｓｉｓｔｅｎｃｙ ｏｆ ＳＩＮＥ ｉｎｓｅｒｔｉｏｎ ｔｏｐｏｌｏｇｙ ａｎｄ ｆｌａｎｋｉｎｇ ｓｅｑｕｅｎｃｅ ｔｒｅｅ： Ｑｕａｎｔｉｆｙｉｎｇ ｒｅｌａｔｉｏｎｓｈｉｐｓ ａｍｏｎｇ ｃｅｔａｒｔｉｏｄａｃｔｙｌｓ． Ｍｏｌ． Ｂｉｏｌ． Ｅｖｏｌ． ２０００； １７：１４１７−１４２４
３１．Ｍａｔｅｒａ Ａ Ｇ， Ｈｅｌｌｍａｎ Ｕ， Ｓｃｈｍｉｄ ＣＷ． Ａ ｔｒａｎｓｐｏｓｉｔｉｏｎａｌｌｙ ａｎｄ ｔｒａｎｓｃｒｉｐｔｉｏｎａｌｌｙ ｃｏｍｐｅｔｅｎｔ Ａｌｕ ｓｕｂｆａｍｉｌｙ． Ｍｏｌ． Ｃｅｌｌ． Ｂｉｏｌ． １９９０； ｉ０： ５４２４−５４３２
３２．Ｍｉｎｎｉｃｋ ＭＦ， Ｓｔｉｌｌｗｅｌｌ ＬＣ， Ｈｅｉｎｅｉｎａｎ ＪＭ， Ｓｔｉｅｇｌｅｒ ＧＬ． Ａ ｈｉｇｈｌｙ ｒｅｐｅｔｉｔｉｖｅ ＤＮＡ ｓｅｑｕｅｎｃｅ ｐｏｓｓｉｂｌｙ ｕｎｉｑｕｅ ｔｏ ｃａｎｉｄｓ． Ｇｅｎｅ １９９２； １１０： ２３５−２３８
３３．Ｍｉｙａｍｏｔｏ ＭＭ． Ｐｅｒｆｅｃｔ ＳＩＮＥｓ ｏｆ ｅｖｏｌｕｔｉｏｎａｒｙ ｈｉｓｔｏｒｙ？ Ｃｕｒｒｅｎｔ Ｂｉｏｌｏｇｙ ２０００；９：８１６−８１９
３４．Ｍｏｃｈｉｚｕｋｉ Ｋ， Ｕｍｅｄａ Ｍ， Ｏｈｔｓｕｂｏ Ｈ， Ｏｈｔｓｕｂｏ Ｅ． Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ａ ｐｌａｎｔ ＳＩＮＥ， ｐ−ＳＩＮＥ１， ｉｎ ｒｉｃｅ ｇｅｎｏｉｎｅｓ． Ｊｐｎ． Ｊ． Ｇｅｎｅｔ． １９９２； ６７： １５５−１６６
３５．Ｍｕｒａｔａ Ｓ， Ｔａｋａｓａｋｉ Ｎ， Ｓａｉｔｏｈ Ｍ， Ｏｋａｄａ Ｎ． Ｄｅｔｅｒｍｉｎａｔｉｏｎ ｏｆ ｔｈｅ ｐｈｙｌｏｇｅｎｅｔｉｃ ｒｅｌａｔｉｏｎｓｈｉｐｓ ａｍｏｎｇ Ｐａｃｉｆｉｃ ｓａｌｍｏｎｉｄｓ ｂｙ ｕｓｉｎｇ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｅｌｅｍｅｎｔｓ （ＳＩＮＥｓ） ａｓ ｔｅｍｐｏｒａｌ ｌａｎｄｍａｒｋｓ ｏｆ ｅｖｏｌｕｔｉｏｎ． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ．， ＵＳＡ １９９３； ９０： ６９９５−６９９９
３６．Ｍｕｒａｔａ Ｓ， Ｔａｋａｓａｋｉ Ｎ， Ｓａｉｔｏｈ Ｍ， Ｔａｃｈｉｄａ Ｈ， Ｏｋａｄａ Ｎ． Ｄｅｔａｉｌｓ ｏｆ ｒｅｔｒｏｐｏｓｉｔｉｏｎａｌ ｇｅｎｏｉｎｅ ｄｙｎａｍｉｃｓ ｔｈａｔ ｐｒｏｖｉｄｅ ａ ｒａｔｉｏｎａｌｅ ｆｏｒ ａ ｇｅｎｅｔｉｃ ｄｉｖｉｓｉｏｎ： ｔｈｅ ｄｉｓｔｉｎｃｔ ｂｒａｎｃｈｉｎｇ ｏｆ ａｌｌ ｔｈｅ Ｐａｃｉｆｉｃ ｓａｌｍｏｎ ａｎｄ ｔｒｏｕｔ （Ｏｎｃｏｒｈｙｎｃｈｕｓ） ｆｒｏｍ ｔｈｅ Ａｔｌａｎｔｉｃ ｓａｌｍｏｎ ａｎｄ ｔｒｏｕｔ （Ｓａｌｍｏ）． Ｇｅｎｅｔｉｃｓ １９９６；１４２：９１５−９２６
３７．Ｎａｇａｈａｓｈｉ ５， Ｅｎｄｏｈ Ｈ， Ｓｕｚｕｋｉ Ｙ， Ｏｋａｄａ Ｎ． Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ａ ｔａｎｄｅｍｌｙ ｒｅｐｅａｔｅｄ ＤＮＡ ｓｅｑｕｅｎｃｅ ｆａｍｉｌｙ ｏｒｉｇｉｎａｌｌｙ ｄｅｒｉｖｅｄ ｂｙ ｒｅｔｒｏｐｏｓｉｔｉｏｎ ｏｆ ｔＲＮＡ（Ｇｌｕ） ｉｎ ｔｈｅ ｎｅｗｔ． Ｊ． Ｍｏｌ． Ｂｉｏｌ． １９９１； ２２２： ３９１−４０４
３８．Ｎｅｉ， Ｍ， Ｋｕｍａｒ Ｓ． Ｍｏｌｅｃｕｌａｒ ｅｖｏｌｕｔｉｏｎ ａｎｄ ｐｈｙｌｏｇｅｎｅｔｉｃｓ． Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ， Ｎｅｗ Ｙｏｒｋ． ２０００．
３９．Ｎｉｋａｉｄｏ Ｍ， Ｒｏｏｎｅｙ ＡＰ， Ｏｋａｄａ Ｎ． Ｐｈｙｌｏｇｅｎｅｔｉｃ ｒｅｌａｔｉｏｎｓｈｉｐｓ ａｍｏｎｇ ｃｅｔａｒｔｉｏｄａｃｔｙｌｓ ｂａｓｅｄ ｏｎ ｉｎｓｅｒｔｉｏｎｓ ｏｆ ｓｈｏｒｔ ａｎｄ ｌｏｎｇ ｉｎｔｅｒｓｐｅｒｓｅｄ ｅｌｅｍｅｎｔｓ： Ｈｉｐｐｏｐｏｔａｍｕｓｅｓ ａｒｅ ｔｈｅ ｃｌｏｓｅｓｔ ｅｘｔａｎｔ ｒｅｌａｔｉｖｅｓ ｏｆ ｗｈａｌｅｓ． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ．， ＵＳＡ １９９９；９６： １０２６１−１０２６６
４０．Ｎｉｋａｉｄｏ Ｍ， Ｍａｔｓｕｎｏ Ｆ， Ｈａｍｉｌｔｏｎ Ｈ， Ｂｒｏｗｎｅｌｌ Ｊｒ． ＲＬ， Ｃａｏ Ｙｉｎ， Ｄｉｎｇ Ｗａｎｇ， Ｚｕｏｙａｎ Ｚｈｕ， Ｓｈｅｄｌｏｃｋ ＡＭ， Ｆｏｒｄｙｃｅ Ｅ， Ｈａｓｅｇａｗａ Ｍ， Ｏｋａｄａ Ｎ． Ｒｅｔｒｏｐｏｓｏｎ ａｎａｌｙｓｉｓ ｏｆ ｍａｊｏｒ ｃｅｔａｃｅａｎ ｌｉｎｅａｇｅｓ： ｔｈｅ ｍｏｎｏｐｈｙｌｙ ｏｆ ｔｏｏｔｈｅｄ ｗｈａｌｅｓ ａｎｄ ｔｈｅ ｐａｒａｐｈｙｌｙ ｏｆ ｒｉｖｅｒ ｄｏｌｐｈｉｎｓ． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ２００１ （ｉｎ ｐｒｅｓｓ）
４１．Ｎｉｋａｉｄｏ Ｍ， Ｈａｒａｄａ Ｍ， Ｃａｏ Ｙ， Ｈａｓｅｇａｗａ Ｍ， Ｏｋａｄａ Ｎ． Ｍｏｎｏｐｈｙｌｅｔｉｃ ｏｒｉｇｉｎ ｏｆ ｔｈｅ ｏｒｄｅｒ Ｃｈｉｒｏｐｔｅｒａ ａｎｄ ｉｔｓ ｐｈｙｌｏｇｅｎｅｔｉｃ ｐｏｓｉｔｉｏｎ ａｍｏｎｇ ｍａｍｍａｌｉａ， ａｓ ｉｎｆｅｒｒｅｄ ｆｒｏｍ ｔｈｅ ｃｏｍｐｌｅｔｅ ｓｅｑｕｅｎｃｅ ｏｆ ｔｈｅ ｍｉｔｏｃｈｏｎｄｒｉａｌ ＤＮＡ ｏｆ ａ Ｊａｐａｎｅｓｅ ｍｅｇａｂａｔ， ｔｈｅ Ｒｙｕｋｙｕ ｆｌｙｉｎｇ ｆｏｘ （Ｐｔｅｒｏｐｕｓ ｄａｓｙｍａｌｌｕｓ）． Ｊ． Ｍｏｌ． Ｅｖｏｌ． ２０００；５１ ：３１８−３２８
４２．Ｏｈｓｈｉｍａ Ｋ， Ｋｏｉｓｈｉ Ｒ， Ｍａｔｓｕｏ Ｍ， Ｏｋａｄａ Ｎ． Ｓｅｖｅｒａｌ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｒｅｐｅｔｉｔｉｖｅ ｅｌｅｍｅｎｔｓ （ＳＩＮＥｓ） ｉｎ ｄｉｓｔａｎｔ ｓｐｅｃｉｅｓ ｍａｙ ｈａｖｅ ｏｒｉｇｉｎａｔｅｄ ｆｒｏｍ ａ ｃｏｍｍｏｎ ａｎｃｅｓｔｒａｌ ｒｅｔｒｏｖｉｒｕｓ： ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ａ ｓｑｕｉｄ ＳＩＮＥ ａｎｄ ａ ｐｏｓｓｉｂｌｅ ｍｅｃｈａｎｉｓｍ ｆｏｒ ｇｅｎｅｒａｔｉｏｎ ｏｆ ｔＲＮＡ−ｄｅｒｉｖｅｄ ｒｅｔｒｏｐｏｓｏｎｓ． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ １９９３；９０：６２６０−６２６４
４３．Ｏｈｓｈｉｍａ Ｋ， Ｏｋａｄａ Ｎ． Ｇｅｎｅｒａｌｉｔｙ ｏｆ ｔｈｅ ｔＲＮＡ ｏｒｉｇｉｎ ｏｆ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｒｅｐｅｔｉｔｉｖｅ ｅｌｅｍｅｎｔｓ （ＳＩＮＥｓ）． Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ｔｈｒｅｅ ｄｉｆｆｅｒｅｎｔ ｔＲＮＡ−ｄｅｒｉｖｅｄ ｒｅｔｒｏｐｏｓｏｎｓ ｉｎ ｔｈｅ ｏｃｔｏｐｕｓ． Ｊ． Ｍｏｌ． Ｂｉｏｌ． １９９４；２４３：２５−３７
４４．Ｏｈｓｈｉｍａ Ｋ， Ｈａｍａｄａ Ｍ， Ｔｅｒａｉ Ｙ， Ｏｋａｄａ Ｎ． Ｔｈｅ ３’ ｅｎｄｓ ｏｆ ｔＲＮＡｄｅｒｉｖｅｄ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｒｅｐｅｔｉｔｉｖｅ ｅｌｅｍｅｎｔｓ ａｒｅ ｄｅｒｉｖｅｄ ｆｒｏｍ ｔｈｅ ３’ ｅｎｄｓ ｏｆ ｌｏｎｇ ｉｎｔｅｒｓｐｅｒｓｅｄ ｒｅｐｅｔｉｔｉｖｅ ｅｌｅｍｅｎｔｓ． Ｍｏｌ． Ｃｅｌｌ． Ｂｉｏｌ． １９９６；１６： ３７５６−３７６４
４５．Ｏｋａｄａ Ｎ． ＳＩＮＥｓ： ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｒｅｐｅａｔｅｄ ｅｌｅｍｅｎｔｓ ｏｆ ｔｈｅ ｅｕｋａｒｙｏｔｉｃ ｇｅｎｏｍｅ． ＴＲＥＥ １９９１ａ；６：３５８−３６１
４６．Ｏｋａｄａ Ｎ． ＳＩＮＥｓ． Ｃｕｒｒ． Ｏｐｉｎ． Ｇｅｎｅｔ． Ｄｅｖ． １９９１ｂ；１：４９８−５０４
４７．Ｏｋａｄａ Ｎ， Ｏｈｓｈｉｍａ Ｋ． Ｅｖｏｌｕｔｉｏｎ ｏｆ ｔＲＮＡ−ｄｅｒｉｖｅｄ ＳＩＮＥｓ． Ｉｎ Ｍａｒａｉａ ＲＪ． ｅｄ； Ｔｈｅ ｉｍｐａｃｔ ｏｆ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｅｌｅｍｅｎｔｓ （ＳＩＮＥｓ） ｏｎ ｔｈｅ ｈｏｓｔ ｇｅｎｏｉｎｅ． Ａｕｓｔｉｎ： ＲＧ Ｌａｎｄｅｓ Ｃｏ． １９９５． ｐ ６２−７９．
４８．Ｏｋａｄａ Ｎ， Ｈａｍａｄａ Ｍ， Ｏｇｉｗａｒａ Ｉ， Ｏｈｓｈｉｍａ Ｋ． ＳＩＮＥｓ ａｎｄ ＬＩＮＥｓ ｓｈａｒｅ ｃｏｍｍｏｎ ３’ ｓｅｑｕｅｎｃｅｓ： ａ ｒｅｖｉｅｗ． Ｇｅｎｅ １９９７；２０５： ２２９−２４３
４９．Ｗａｄｄｅｌ ＰＪ， Ｏｋａｄａ Ｎ， Ｈａｓｅｇａｗａ Ｍ Ｔｏｗａｒｄｓ ｒｅｓｏｌｖｉｎｇ ｔｈｅ ｉｎｔｅｒｏｒｄｉｎａｌ ｒｅｌａｔｉｏｎｓｈｉｐｓ ｏｆ ｐｌａｃｅｎｔａｌ ｍａｍｍａｌｓ． Ｓｙｓｔ． Ｂｉｏｌ． １９９９；４８：１−５
５０．Ｒｏｇｅｒｓ Ｊ． Ｒｅｔｒｏｐｏｓｏｎｓ ｄｅｆｉｎｅｄ． Ｎａｔｕｒｅ １９８３；３０１：４６０
５１．Ｒｏｋａｓ Ａ， Ｈｏｌｌａｎｄ ＰＷＨ． Ｒａｒｅ ｍｏｌｅｃｕｌａｒ ｃｈａｎｇｅｓ ａｓ ａ ｔｏｏｌ ｆｏｒ ｐｈｙｌｏｇｅｎｅｔｉｃｓ． ＴＲＥＥ （ｉｎ ｐｒｅｓｓ）
５２．Ｓａｋａｇａｍｉ Ｍ， Ｏｈｓｈｉｍａ Ｋ， Ｍｕｋｏｙａｍａ Ｈ， Ｙａｓｕｅ Ｈ， Ｏｋａｄａ Ｎ． Ａ ｎｏｖｅｌ ｔＲＮＡ ｓｐｅｃｉｅｓ ａｓ ａｎ ｏｒｉｇｉｎ ｏｆ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｒｅｐｅｔｉｔｉｖｅ ｅｌｅｍｅｎｔｓ （ＳＩＮＥｓ）． Ｅｑｕｉｎｅ ＳＩＮＥｓ ｍａｙ ｈａｖｅ ｏｒｉｇｉｎａｔｅｄ ｆｒｏｍ ｔＲＮＡ（Ｓｅｒ）． Ｊ． Ｍｏｌ． Ｂｉｏｌ． １９９４；２３９：７３１−７３５
５３．Ｓｃｈｍｉｄ Ｃ． Ａｌｕ： ｓｔｒｕｃｔｕｒｅ， ｏｒｉｇｉｎ， ｅｖｏｌｕｔｉｏｎ， ｓｉｇｎｉｆｉｃａｎｃｅ ａｎｄ ｆｕｎｃｔｉｏｎ ｏｆ ｏｎｅ−ｔｅｎｔｈ ｏｆ ｈｕｍａｎ ＤＮＡ． Ｐｒｏｇ． Ｎｕｃｌ． Ａｃｉｄ Ｒｅｓ． ａｎｄ Ｍｏｌ． Ｂｉｏｌ． １９９６；５３：２８３−３１９
５４．Ｓｃｈｍｉｄ ＣＷ， Ｍａｒａｉａ Ｒ． Ｔｒａｎｓｃｒｉｐｔｉｏｎａｌ ｒｅｇｕｌａｔｉｏｎ ａｎｄ ｔｒａｎｓｐｏｓｉｔｉｏｎａｌ ｓｅｌｅｃｔｉｏｎ ｏｆ ａｃｔｉｖｅ ＳＩＮＥ ｓｅｑｕｅｎｃｅｓ． Ｃｕｒｒ． Ｏｐｉｎ． Ｇｅｎｅｔ． Ｄｅｖｅｌ． １９９２；２： ８７４−８８２
５５．Ｓｃｈｍｉｔｚ Ｊ， Ｏｈｘｎｅ Ｍ， Ｚｉｓｃｈｌｅｒ Ｈ． ＳＩＮＥ ｉｎｓｅｒｔｉｏｎｓ ｉｎ ｃｌａｄｉｓｔｉｃ ａｎａｌｙｓｅｓ ａｎｄ ｔｈｅ ｐｈｙｌｏｇｅｎｅｔｉｃ ａｆｆｉｌｉａｔｉｏｎｓ ｏｆ Ｔａｒｓｉｕｓ ｂａｎｃａｎｕｓ ｔｏ ｏｔｈｅｒ Ｐｒｉｍａｔｅｓ．Ｇｅｎｅｔｉｃｓ ２００１ （ｉｎ ｐｒｅｓｓ）
５６．Ｓｈｅｄｌｏｃｋ， ＡＭ．， Ｏｋａｄａ Ｎ． ＳＩＮＥ ｉｎｓｅｒｔｉｏｎｓ： Ｐｏｗｅｒｆｕｌ ｔｏｏｌｓ ｆｏｒ ｍｏｌｅｃｕｌａｒ ｓｙｓｔｅｍａｔｉｃｓ． ＢｉｏＥｓｓａｙｓ ２０００； ２２：１４８−１６０
５７．Ｓｈｅｄｌｏｃｋ ＡＭ， Ｍｉｌｉｎｋｏｖｉｔｃｈ ＭＣ， Ｏｋａｄａ Ｎ． ＳＩＮＥ ｅｖｏｌｕｔｉｏｎ， ｍｉｓｓｉｎｇ ｄａｔａ， ａｎｄ ｔｈｅ ｏｒｉｇｉｎ ｏｆ ｗｈａｌｅｓ． Ｓｙｓｔ． Ｂｉｏｌ． ２０００； ４９：８０８−８１７
５８．Ｓｈｅｄｌｏｃｋ ＡＭ， Ｔａｋａｈａｓｈｉ Ｋ， Ｏｋａｄａ Ｎ． ＳＩＮＥｓ ｏｆ ｓｐｅｃｉａｔｉｏｎ： ｔｒａｃｋｉｎｇ ｔｈｅ ｓｏｒｔｉｎｇ ｏｆ ｌｉｎｅａｇｅｓ ｗｉｔｈ ｒｅｔｒｏｐｏｓｏｎｓ． ＴＲＥＥ ２００１
５９．Ｓｈｅｎ ＭＲ， Ｂａｔｚｅｒ ＭＡ， Ｄｅｉｎｉｎｇｅｒ ＰＬ． Ｅｖｏｌｕｔｉｏｎ ｏｆ ｔｈｅ Ｍａｓｔｅｒ Ａｌｕ Ｇｅｎｅ （ｓ）． Ｊ． Ｍｏｌ． Ｅｖｏｌ． １９９１；３３： ３１１−３２０
６０．Ｓｈｅｒｒｙ ＳＴ， Ｈａｒｐｅｎｄｉｎｇ ＨＣ， Ｂａｔｚｅｒ ＭＡ， Ｓｔｏｎｅｋｉｎｇ Ｍ． Ａｌｕ ｅｖｏｌｕｔｉｏｎ ｉｎ ｈｕｍａｎ ｐｏｐｕｌａｔｉｏｎｓ： Ｕｓｉｎｇ ｔｈｅ ｃｏａｌｅｓｃｅｎｔ ｔｏ ｅｓｔｉｍａｔｅ ｅｆｆｅｃｔｉｖｅ ｐｏｐｕｌａｔｉｏｎ ｓｉｚｅ． Ｇｅｎｅｔｉｃｓ １９９７； １４７：１９７７−１９８２
６１．Ｓｈｉｍａｍｕｒａ Ｍ， Ｙａｓｕｅ Ｈ， Ｏｈｓｈｉｍａ Ｋ， Ａｂｅ Ｈ， Ｋａｔｏ Ｈ， Ｋｉｓｈｉｒｏ Ｔ， Ｇｏｔｏ Ｍ， Ｍｕｎｅｃｈｉｋａ Ｉ， Ｏｋａｄａ Ｎ． Ｍｏｌｅｃｕｌａｒ ｅｖｉｄｅｎｃｅ ｆｒｏｍ ｒｅｔｒｏｐｏｓｏｎｓ ｔｈａｔ ｗｈａｌｅｓ ｆｏｒｍ ａ ｄａｄｅ ｗｉｔｈｉｎ ｅｖｅｎ−ｔｏｅｄ ｕｎｇｕｌａｔｅｓ． Ｎａｔｕｒｅ １９９７；３８８： ６６６−６７０
６２．Ｓｈｉｍａｍｕｒａ Ｍ， Ａｂｅ Ｈ， Ｎｉｋａｉｄｏ Ｍ， Ｏｈｓｈｉｍａ Ｋ， Ｏｋａｄａ Ｎ． Ｇｅｎｅａｌｏｇｙ ｏｆ ｆａｍｉｌｉｅｓ ｏｆ ＳＩＮＥｓ ｉｎ ｃｅｔａｃｅａｎ ａｎｄ ａｒｔｉｏｄａｃｔｙｌｓ： Ｔｈｅ ｐｒｅｓｅｎｃｅ ｏｆ ａ ｈｕｇｅ ｓｕｐｅｒｆａｍｉｌｙ ｏｆ ｔＲＮＡＧ１ｕ−ｄｅｒｉｖｅｄ ｆａｍｉｌｉｅｓ ｏｆ ＳＩＮＥｓ． Ｍｏｌ． Ｂｉｏｌ． Ｅｖｏｌ． １９９９；１６：１０４６−１０６０
６３．Ｓｍｉｔ ＡＦＡ， Ｔｏｔｈ Ｇ， Ｒｉｇｇｓ ＡＤ， Ｊｕｒｋａ Ｊ． Ａｎｃｅｓｔｒａｌ， ｍａｍｍａｌｉａｎ−ｗｉｄｅ ｓｕｂｆａｍｉｌｉｅｓ ｏｆ ＬＩＮＥ−ｉ ｒｅｐｅｔｉｔｉｖｅ ｓｅｑｕｅｎｃｅｓ． Ｊ． Ｍｏｌ． Ｅｖｏｌ． １９９５； ２４６：４０１−４１７
６４．Ｓｍｉｔ ＡＦＡ， Ｒｉｇｇｓ ＡＤ． ＭＩＲ５ ａｒｅ ｃｌａｓｓｉｃ， ｔＲＮＡ−ｄｅｒｉｖｅｄ ＳＩＮＥｓ ｔｈａｔ ａｍｐｌｉｆｉｅｄ ｂｅｆｏｒｅ ｔｈｅ ｍａｍｍａｌｉａｎ ｒａｄｉａｔｉｏｎ． Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． １９９５； ２３：９８−１０２
６５．Ｓｐｒｉｎｚｌ Ｍ． Ｈａｒｔｍａｎｎ Ｔ， Ｍｅｉｓｓｎｅｒ Ｆ， Ｍｏｌｌ Ｊ， Ｖｏｒｄｅｒｗｉｉｌｂｅｃｋｅ Ｔ． Ｃｏｍｐｉｌａｔｉｏｎ ｏｆ ｔＲＮＡ ｓｅｑｕｅｎｃｅｓ ａｎｄ ｓｅｑｕｅｎｃｅｓ ｏｆ ｔＲＮＡ ｇｅｎｅｓ． Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． １９８７；１５：ｒ５３−ｒ１８８
６６．Ｓｔｏｎｅｋｉｎｇ Ｍ． ｅｔ ａｌ． Ａｌｕ ｉｎｓｅｒｔｉｏｎ ｐｏｌｙｍｏｒｐｈｉｓｍｓ ａｎｄ ｈｕｍａｎ ｅｖｏｌｕｔｉｏｎ： Ｅｖｉｄｅｎｃｅ ｆｏｒ ａ ｌａｒｇｅｒ ｐｏｐｕｌａｔｉｏｎ ｓｉｚｅ ｉｎ Ａｆｒｉｃａ． Ｇｅｎｏｍｅ Ｒｅｓ． １９９７；７：１０６１−１０７１
６７．Ｓｕｒｚｙｃｋｉ ＳＡ， Ｂｅｌｋｎａｐ ＷＲ． Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ｒｅｐｅｔｉｔｉｖｅ ＤＮＡ ｅｌｅｍｅｎｔｓ ｉｎ Ａｒａｂｉｄｏｐｓｉｓ． Ｊ． Ｍｏｌ． Ｂｉｏｌ． １９９９；４８：６８４−６９１
６８．Ｔａｋａｈａｓｈｉ Ｋ， Ｔｅｒａｉ Ｙ， Ｎｉｓｈｉｄａ Ｍ， Ｏｋａｄａ Ｎ． Ａ ｎｏｖｅｌ ｆａｍｉｌｙ ｏｆ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｒｅｐｅｔｉｔｉｖｅ ｅｌｅｍｅｎｔｓ （ＳＩＮＥｓ） ｆｒｏｍ ｃｉｃｈｌｉｄｓ： ｔｈｅ ｐａｔｔｅｒｎ ｏｆ ｉｎｓｅｒｔｉｏｎ ｏｆ ＳＩＮＥｓ ａｔ ｏｒｔｈｏｌｏｇｏｏｕｓ ｌｏｃｉ ｓｕｐｐｏｒｔ ｔｈｅ ｐｒｏｐｏｓｅｄ ｍｏｎｏｐｈｙｌｙ ｏｆ ｆｏｕｒ ｍａｊｏｒ ｇｒｏｕｐｓ ｏｆ ｃｉｃｈｌｉｄ ｆｉｓｈｅｓ ｉｎ Ｌａｋｅ Ｔａｎｇａｎｙｉｋａ． Ｍｏｌ． Ｂｉｏｌ． Ｅｖｏｌ． １９９８；１５：３９１−４０７
６９．Ｔａｋａｓａｋｉ Ｎ， Ｍｕｒａｔａ ５， Ｓａｉｔｏｈ Ｍ， Ｋｏｂａｙａｓｈｉ Ｔ， Ｐａｒｋ Ｌ， Ｏｋａｄａ Ｎ． Ｓｐｅｃｉｅｓ−ｓｐｅｃｉｆｉｃ ａｍｐｌｉｆｉｃａｔｉｏｎ ｏｆ ｔＲＮＡ−ｄｅｒｉｖｅｄ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｒｅｐｅｔｉｔｉｖｅ ｅｌｅｍｅｎｔｓ （ＳＩＮＥｓ） ｂｙ ｒｅｔｒｏｐｏｓｉｔｉｏｎ： Ａ ｐｒｏｃｅｓｓ ｏｆ ｐａｒａｓｉｔｉｚａｔｉｏｎ ｏｆ ｅｎｔｉｒｅ ｇｅｎｏｉｎｅｓ ｄｕｒｉｎｇ ｔｈｅ ｅｖｏｌｕｔｉｏｎ ｏｆ ｓａｌｍｏｎｉｄｓ． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ １９９４；９１： １０１５３−１０１５７
７０．Ｔａｋａｓａｋｉ Ｎ， Ｐａｒｋ Ｌ， Ｋａｅｒｉｙａｉｎａ Ｍ， Ｇｈａｒｒｅｔｔ ＡＪ， Ｏｋａｄａ Ｎ． Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ｓｐｅｃｉｅｓ−ｓｐｅｃｉｆｉｃａｌｌｙ ａｍｐｌｉｆｉｅｄ ＳＩＮＥｓ ｉｎ ｔｈｒｅｅ ｓａｌｍｏｎｉｄ ｓｐｅｃｉｅｓ − ｃｈｕｍ ｓａｌｍｏｎ， ｐｉｎｋ ｓａｌｍｏｎ， ａｎｄ ｋｏｋａｎｅｅ： ｔｈｅ ｌｏｃａｌ ｅｎｖｉｒｏｍｅｎｔ ｏｆ ｔｈｅ ｇｅｎｏｉｎｅ ｍａｙ ｂｅ ｉｍｐｏｒｔａｎｔ ｆｏｒ ｔｈｅ ｇｅｎｅｒａｔｉｏｎ ｏｆ ａ ｄｏｍｉｎａｎｔ ｓｏｕｒｃｅ ｇｅｎｅ ａｔ ａ ｎｅｗｌｙ ｒｅｔｒｏｐｏｓｅｄ ｌｏｃｕｓ． Ｊ． Ｍｏｌ． Ｅｖｏｌ． １９９６；４２：１０３−１０６
７１．Ｔｅｒａｉ Ｙ， Ｔａｋａｈａｓｈｉ Ｋ， Ｏｋａｄａ Ｎ． ＳＩＮＥ Ｃｏｕｓｉｎｓ： Ｔｈｅ ３’ ｅｎｄ ｔａｉｌｓ ｏｆ ｔｈｅ ｔｗｏ ｏｌｄｅｓｔ ａｎｄ ｄｉｓｔａｎｔｌｙ ｒｅｌａｔｅｄ ｆａｍｉｌｉｅｓ ｏｆ ＳＩＮＥｓ ａｒｅ ｄｅｓｃｅｎｄｅｄ ｆｒｏｍ ｔｈｅ ３’ ｅｎｄｓ ｏｆ ＬＩＮＥｓ ｗｉｔｈ ｔｈｅ ｓａｍｅ ｇｅｎｅｏｌｏｇｉｃａｌ ｏｒｉｇｉｎ． Ｍｏｌ． Ｂｉｏｌ． Ｅｖｏｌ． １９９８；１５： １４６０−１４７１
７２．Ｕｎｓａｌ Ｋ， Ｍｏｒｇａｎ ＧＴ． Ａ ｎｏｖｅｌ ｇｒｏｕｐ ｏｆ ｆａｍｉｌｉｅｓ ｏｆ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｒｅｐｅｔｉｔｉｖｅ ｅｌｅｍｅｎｔｓ （ＳＩＮＥｓ） ｉｎ Ｘｅｎｏｐｕｓ： ｅｖｉｄｅｎｃｅ ｏｆ ａ ｓｐｅｃｉｆｉｃ ｔａｒｇｅｔ ｓｉｔｅ ｆｏｒ ＤＮＡ−ｍｅｄｉａｔｅｄ ｔｒａｎｓｐｏｓｉｔｉｏｎ ｏｆ ｉｎｖｅｒｔｅｄ− ｒｅｐｅａｔ ＳＩＮＥｓ． Ｊ． Ｍｏｌ． Ｂｉｏｌ． １９９５； ２４８：８１２−８２３
７３．ｖａｎ ｄｅｒ Ｖｌｕｇｔ ＨＨＪ， Ｌｅｎｓｔｒａ ＪＡ． ＳＩＮＥ ｅｌｅｍｅｎｔｓ ｏｆ ｃａｒｎｉｖｏｒｅｓ． Ｍａｍｍａｌ． Ｇｅｎｏｍｅ １９９５；６：４９−５１
７４．Ｗｅｉｎｅｒ Ａ， Ｄｅｉｎｉｎｇｅｒ ＰＬ， Ｅｆｓｔｒａｔｉａｄｉｓ Ａ． Ｎｏｎｖｉｒａｌ ｒｅｔｒｏｐｏｓｏｎｓ： ｇｅｎｅｓ， ｐｓｅｕｄｏｇｅｎｅｓ， ａｎｄ ｔｒａｎｓｐｏｓａｂｌｅ ｅｌｅｍｅｎｔｓ ｇｅｎｅｒａｔｅｄ ｂｙ ｔｈｅ ｒｅｖｅｒｓｅ ｆｌｏｗ ｏｆ ｇｅｎｅｔｉｃ ｉｎｆｏｒｍａｔｉｏｎ． Ａｎｎ． Ｒｅｖ． Ｂｉｏｃｈｅｍ． １９８６；５５： ６３１−６６１
７５．Ｙｏｓｈｉｏｋａ Ｙ， Ｍａｔｓｕｍｏｔｏ ５， Ｋｏｊｉｉｎａ ５， Ｏｈｓｈｉｍａ Ｋ， Ｏｋａｄａ Ｎ， Ｍａｃｈｉｄａ Ｙ． Ｍｏｌｅｃｕｌａｒ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ａ ｓｈｏｒｔ ｉｎｔｅｒｓｐｅｒｓｅｄ ｒｅｐｅｔｉｔｉｖｅ ｅｌｅｍｅｎｔ ｆｒｏｍ ｔｏｂａｃｃｏ ｔｈａｔ ｅｘｈｉｂｉｔｓ ｓｅｑｕｅｎｃｅ ｈｏｍｏｌｏｇｙ ｔｏ ｓｐｅｃｉｆｉｃ ｔＲＮＡ５． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ １９９３； ９０：６５６２−６５６６
【００８７】
【配列表】

【図面の簡単な説明】
【図１】ＳＩＮＥのレトロポジションに関する一般ダイアグラム。
【図２】図２（Ａ）はマスター遺伝子モデルを表し、そして図２（Ｂ）は多数源遺伝子モデルを表す。
【図３】ＳＩＮＥの進化の指標としての有用性を示す。
【図４】種Ａ〜Ｄの系統樹モデルを表す。図４（Ｃ）はＰＣＲパターンを示す。
【図５】選択された動物種由来の全ゲノムＤＮＡからの転写産物のいくつかのパターン例である。
【図６】ＣＨＲ−２ ＳＩＮＥのｔＲＮＡ様構造を示す。
【図７】図７（Ａ）はＣＨＲ−２ ＳＩＮＥのｔＲＮＡ様二次構造を示し、そして図７（Ｂ）はヒトｔＲＮＡ Ｇｌｕの二次構造を示す。
【図８】ＣＨＲ−２のいくつかの配列アラインメントからのＣＨＲ−２ ＳＩＮＥのコンセンサス配列の構築を表す。
【図９】ｔの時に増幅された古いＳＩＮＥと、ｕの時に増幅された若いＳＩＮＥを含む系統分類樹。
【図１０】アラインメントによるＳＩＮＥファミリーのサブファミリーを表す特徴的なヌクレオチド又は可能な欠失の同定を表す。
【図１１】ＣＨＲ−２ ＳＩＮＥファミリーのサブファミリーのコンセンサス配列のアラインメントを示す。
【図１２】ＣＤ，ＣＤＯ，及び他のサブファミリーに特異的なプローブを、それぞれ、用いたドット−ハイブリダイゼーション実験の結果を示す。
【図１３】フランキングＳＩＮＥ ＰＣＲの原理を模式的に示す。
【図１４】フランキングＰＣＲ結果の１例を示す。また、図１４（Ｂ）と（Ｃ）に２つの異なるプローブを用いた同一フィルターを用いて行ったハイブリダイゼーション実験結果を同時に示す。
【図１５】哺乳動物の系統発生樹を示す。
【図１６】ＳＩＮＥ法における実験の流れを示す。
【図１７】分子統計学的研究から提唱された系統樹（歯鯨類の単系統性の問題をめぐる論争）を示す。
【図１８】ＣＨＲ−２各サブファミリーのコンセンサス配列のアラインメントを示す。
【図１９】鯨目に共通にＳＩＮＥの挿入があったことを示す遺伝子座Ｂａｎｄｏ１とＳｐ３１６のバンドパターンを示す。
【図２０】遺伝子座Ｂａｎｄｏ１のアラインメントの結果を示す。
【図２１】遺伝子座Ｂａｎｄｏ１のアラインメントの結果を示す。
【図２２】遺伝子座Ｂａｎｄｏ１のアラインメントの結果を示す。
【図２３】遺伝子座Ｓｐ３１６のアラインメントの結果を示す。
【図２４】イルカ上科４種（マイルカ科：コビレゴンドウ、バンドウイルカ；ネズミイルカ科：イシイルカ；イッカク科：イッカク）に特異的なＣＤＯの挿入があったことを示す遺伝子座Ｍａｇｏ１９のバンドパターンを示す。この遺伝子座はイルカ上科の単系統性を強く支持する。
【図２５】遺伝子座Ｍａｇｏ１９のアラインメントの結果を示す。
【図２６】遺伝子座Ｍａｇｏ１９のアラインメントの結果を示す。
【図２７】イルカ上科４種、南米カワイルカ２種アマゾンカワイルカ、ラプラタカワイルカ及びヨウスコウカワイルカにＣＤＯの挿入があったことを示す遺伝子座Ｉｓｉ１４， Ｉｓｉ３６， Ｉｓｉ３８のバンドパターンを示す。
【図２８】イルカ上科４種、南米カワイルカ２種アマゾンカワイルカ、ラプラタカワイルカ及びヨウスコウカワイルカにＣＤＯの挿入があったことを示す遺伝子座Ｍａｇｏ２４， Ｍａｇｏ２６， Ｍａｇｏ３２のバンドパターンを示す。
【図２９】遺伝子座Ｉｓｈｉ１４のアラインメントの結果を示す。
【図３０】遺伝子座Ｉｓｈｉ３６のアラインメントの結果を示す。
【図３１】遺伝子座Ｉｓｈｉ３６のアラインメントの結果を示す。
【図３２】遺伝子座Ｉｓｈｉ３８のアラインメントの結果を示す。
【図３３】遺伝子座Ｉｓｈｉ３８のアラインメントの結果を示す。
【図３４】遺伝子座Ｍａｇｏ２４のアラインメントの結果を示す。
【図３５】遺伝子座Ｍａｇｏ２４のアラインメントの結果を示す。
【図３６】遺伝子座Ｍａｇｏ２６のアラインメントの結果を示す。
【図３７】遺伝子座Ｍａｇｏ２６のアラインメントの結果を示す。
【図３８】遺伝子座Ｍａｇｏ３２のアラインメントの結果を示す。
【図３９】遺伝子座Ｍａｇｏ３２のアラインメントの結果を示す。
【図４０】Ｄｅｌｐｈｉｎｉｄａとアカボウクジラ科に属する２種ツチクジラ、オオギハクジラに特異的なＣＤＯの挿入があったことを示す遺伝子座Ｍａｇｏ８， Ｍａｇｏ１３のバンドパターンを示す。
【図４１】遺伝子座Ｍａｇｏ８のアラインメントの結果を示す。
【図４２】遺伝子座Ｍａｇｏ１３のアラインメントの結果を示す。
【図４３】Ｄｅｌｐｈｉｎｉｄａ、アカボウクジラ科及びガンジスカワイルカに特異的なＣＤＯの挿入があったことを示す遺伝子座Ｍａｇｏ２１， Ｍａｇｏ２２のバンドパターンを示す。
【図４４】遺伝子座Ｍａｇｏ２１のアラインメントの結果を示す。
【図４５】遺伝子座Ｍａｇｏ２２のアラインメントの結果を示す。
【図４６】遺伝子座Ｍａｇｏ２２のアラインメントの結果を示す。
【図４７】遺伝子座Ｍａｇｏ２２のアラインメントの結果を示す。
【図４８】マッコウクジラも含めた歯鯨亜目全ての種で共通にＣＤの挿入があったことを示す遺伝子座Ｓｐｅｒｍ８， Ｓｐｅｒｍ２８， Ｓｐｅｒｍ４７のバンドパターンを示す。
【図４９】遺伝子座Ｓｐｅｒｍ８のアラインメントの結果を示す。
【図５０】遺伝子座Ｓｐｅｒｍ８のアラインメントの結果を示す。
【図５１】遺伝子座Ｓｐｅｒｍ２８のアラインメントの結果を示す。
【図５２】遺伝子座Ｓｐｅｒｍ４７のアラインメントの結果を示す。
【図５３】南米カワイルカ２種アマゾンカワイルカ、ラプラタカワイルカにおいて共通にＣＤＯの挿入があったことを示す遺伝子座Ａｍｚ１３， Ｂａｎｄｏ１のバンドパターンを示す。
【図５４】遺伝子座Ａｍｚ１３のアラインメントの結果を示す。
【図５５】アマゾンカワイルカに特異的にＣＤＯの挿入があったことを示す遺伝子座Ａｍｚ１１のバンドパターンを示す。
【図５６】遺伝子座Ａｍｚ１１のアラインメントの結果を示す。
【図５７】アカボウクジラ科に特異的にＣＤＯの挿入があったことを示す遺伝子座Ｔｕｔｉ２４， Ｔｕｔｉ３５のバンドパターンを示す。
【図５８】遺伝子座Ｔｕｔｉ２４のアラインメントの結果を示す。
【図５９】遺伝子座Ｔｕｔｉ３５のアラインメントの結果を示す。
【図６０】遺伝子座Ｔｕｔｉ３５のアラインメントの結果を示す。
【図６１】マッコウクジラ上科３種にＣＤＯの挿入があったことを示す遺伝子座Ｓｐ９、及びマッコウクジラ科に特異的なＳｐ２のバンドパターンを示す。
【図６２】遺伝子座Ｓｐ２のアラインメントの結果を示す。
【図６３】遺伝子座Ｓｐ２のアラインメントの結果を示す。
【図６４】遺伝子座Ｓｐ９のアラインメントの結果を示す。
【図６５】ナガスクジラ科（又は髭鯨）に特異的なＣＤの挿入があったことを示す遺伝子座Ｈｕｍｐ２０， Ｈｕｍｐ２０３のバンドパターンを示す。
【図６６】遺伝子座Ｈｕｍｐ２０のアラインメントの結果を示す。
【図６７】遺伝子座Ｈｕｍｐ２０のアラインメントの結果を示す。
【図６８】遺伝子座Ｈｕｍｐ２０のアラインメントの結果を示す。
【図６９】遺伝子座Ｈｕｍｐ２０３のアラインメントの結果を示す。
【図７０】ＳＩＮＥの挿入パターンをまとめたマトリックスを示す。
【図７１】図７０に示すマトリックスに基づき推定される鯨目内部の系統発生樹を示す。
【図７２】分子統計学的解析の結果（限界）を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for determining the species of an animal by the SINE method. More particularly, the present invention relates to a method for discriminating the species of mammals including cetaceans using the SINE subfamily.
[0002]
[Prior art]
The automation of DNA sequencing and molecular cloning techniques has dramatically increased access to an organism's genome. In addition, the flood of genomic digital information has attracted many biologists to computer-aided bioinformatics. The results of a comprehensive effort, such as the Human Genome Project, have shown in great detail what nucleic acid regeneration studies have revealed about 30 years ago. That is, the vast majority of eukaryotic genomes do not encode specific protein products and are composed of repetitive elements with no apparent function (Kazazian et al., 1998). As a result of genome analysis, the so-called "junk yard", which does not code for proteins, has become a new "jungle" of dynamic molecules filled with active elements such as DNA type transposable elements and RNA type transposable elements. Be recognized. Retroposons are interesting ubiquitous repetitive sequences that are being considered as taxonomic tools for phylogenetic reconstruction and population analysis, and are members of the RNA transposable element (Brosius, 1991; Shedlock and Okada, 2000). Therefore, retroposon diagnosis provides an important bridge between relevant sub-disciplines of evolutionary biology at the individual or species level.
[0003]
The term "retroposition" refers to how such elements move between a parent locus and a target locus in the genome by an RNA intermediate (Rogers, 1983). This copy and paste process creates a reversal of genetic information from RNA to chromosomal DNA (Weiner, et al., 1986, Schmid, 1996). It differs from DNA-type transposons, such as the mariner and P elements, which jump around the chromosome in a manner that leaves no original copy at the parent locus, ie, in a cut-and-paste manner (Clark and Kidwell, 1997; Hartl et al, 1997). An important feature used to classify retroposons is whether they encode reverse transcriptase (RTase), an enzyme essential for self-amplification (Okada, 1991; Eickbush, 1994). Among non-self-amplifying retroposons, short interspersed elements (SINEs) are considered to be extremely useful for the study of eukaryotic taxonomy because of their large number in the genome. . SINEs range in size from 70-500 bp and fall into two categories, those derived from tRNA and those derived from 7SLRNA. The SINE from 7SLRNA contains only two SINE families, the Alu family of primates and the B1 family of rodents. All other SINEs examined so far have been shown to be derived from tRNA. LINEs that encode RTase for amplification also fall into two categories. That is, a mammalian L1 that requires only a simple polyA sequence for reverse transcription without the need for a specific sequence for amplification, and a strict 3 ′ end to be recognized by RTase for amplification. Other categories, including most LINEs, that require a unique sequence motif. Most non-mammalian SINEs and LINEs share the same 3 'tail sequence, and their presence allows SINE to be amplified by the LINE encoded RTase (Ohshima et al, 1997; Okada). et al, 1997; Terai et al., 1998). In the case of mammals, SINE, including those from tRNA and 7SL RNA, appears to be amplified with the help of RTase encoded by mammalian L1. Therefore, in this case, the above conserved sequence motif is not observed at the 3 'tail of SINE. A general diagram for the SINE retroposition is shown in FIG.
[0004]
The fact that a retro position always occurs in one direction and 10 of SINE⁴The fact that these copies are scattered throughout the host genome highlights that SINE insertion analysis is a powerful new approach to molecular taxonomy. This SINE insertion analysis complements the analysis of other forms with character data, most notably DNA sequence and morphology (eg, Murata et al, 1993, Shimamura et al., 1997, Takahashi et al, 1998). Nikaido et al, 1999). Papers on SINE evolution as a tool for taxonomy and their importance are available (Weiner et al, 1996, Deininger and Batzer, 1993, Schmid, 1996, Rokas and Holland, 2000; Shedlock and Okadah, 2000, Shedlock and Okadah, 2000; 2000).
[0005]
By the way, in discriminating animal species including whales, after amplifying a specific region of the mitochondrial gene by PCR, electrophoresing them on an agarose gel and confirming the amplification, and then sequencing using a sequencing method Species have been determined by determining the sequence and using the difference in the sequence as an index. Due to the high cost of the sequencing method equipment, most of these analyzes are currently performed by specialized companies. Therefore, in order to determine the species of an enormous number of individuals, the number of analyzes is naturally enormous, and the cost is enormous. Furthermore, the analysis of the base sequence still has insufficient and uncertain aspects as described below, and their interpretation is often regarded as a problem.
[0006]
[Problems to be solved by the invention]
Therefore, there is still a need for an assay method for simply, rapidly and inexpensively discriminating animal species including whales without using nucleotide sequence analysis.
[0007]
[Means for Solving the Problems]
Recently, the inventor of the present application has found that SINE can be used not only for the study of phylogeny and taxonomy as described above, but also for a simple, rapid and inexpensive test method for discriminating the species of animals including whales. discovered. As a result of intensive experiments on a wide range of samples, the present inventors have proved their reliability and completed the present invention. That is, as described in detail below, if the SINE insertions are common, it is possible to phylogenetically reveal that certain commonly inserted species are monophylic to each other. If the insertion of the SINE family or subfamily is unique within a particular species, it can be seen that the presence or absence of the insertion can be used for species discrimination. Methods for isolating and identifying the SINE subfamily sequence specific to such species are also within the scope of the present invention.
Conventionally, phylogenetic trees have been created using the SINE family, but this has been done without using the SINE family to classify as a subfamily. Since the SINE that enables species identification cannot be performed without a recently amplified SINE, it is necessary to specifically isolate the recently amplified subfamily. This method submits the method.
[0008]
In one embodiment of the present invention, there is provided a method for determining an animal species by the SINE method, comprising the following steps:
Creating a genomic DNA library from one or more animal species to be identified;
Isolating from each said library a clone containing an orthologous locus in which at least one of said animal species has specifically inserted the SINE family or subfamily;
Using a set of PCR primers that anneal to flanking sequences flanking the SINE family or subfamily at the locus, PCR amplify the sequence at the locus for each of the animal species; Gel species of the PCR product to determine the species of the animal by the presence or absence of a band indicating the presence of the SINE family or subfamily insertion locus;
The method is provided, comprising:
The SINE family or subfamily can be a DNA having a sequence selected from the group consisting of SEQ ID NOs: 3 to 7 or a SINE DNA that hybridizes with the above DNA under stringent conditions. For example, the SINE subfamily is a DNA having the CD sequence shown in SEQ ID NO: 6 or the CDO sequence shown in SEQ ID NO: 7.
[0009]
In another aspect of the present invention, there is provided a SINE family or subfamily DNA having a sequence selected from the group consisting of SEQ ID NOs: 3 to 7, or a SINE DNA that hybridizes with the above DNA under stringent conditions.
Whether the orthologous locus is Bandol corresponding to the sequence shown in any one of SEQ ID NOs: 8 to 21, Sp316 corresponding to the sequence shown in any one of SEQ ID NOs: 22 to 35, or SEQ ID NO: 36 to 46 is Mago19 corresponding to the sequence shown in any one of SEQ ID NOs: 47 to 51, is Ishi14 corresponding to the sequence shown in any one of SEQ ID NOs: 47 to 51, or is equivalent to the sequence shown in any one of SEQ ID NOS: 52 to 65 Ishi36, Ishi38 corresponding to the sequence shown in any one of SEQ ID NOs: 66 to 79, Mago24 corresponding to the sequence shown in any one of SEQ ID NOs: 211 to 224, or SEQ ID NO: 225 238, or Mago26 corresponding to the sequence shown in any one of SEQ ID NOs: 80 to 93. 2, Mago8 corresponding to the sequence shown in any one of SEQ ID NOs: 94 to 98, Mago13 corresponding to the sequence shown in any one of SEQ ID NOs: 99 to 112, or SEQ ID NOS: 113 to 117 Or Mago22 corresponding to the sequence shown in any one of SEQ ID NOs: 239 to 252, or corresponds to the sequence shown in any one of SEQ ID NOS: 118 to 129 Sperm8, Sperm28 corresponding to the sequence shown in any one of SEQ ID NOS: 130 to 135, Sperm47 corresponding to the sequence shown in any one of SEQ ID NOS: 136 to 140, SEQ ID NOS: 141 to 145 Amz13 corresponding to the sequence shown in any one of SEQ ID NOs: 146 to 151, or Amti11, Tuti24 corresponding to the sequence shown in any one of SEQ ID NOs: 152 to 156, Tuti35 corresponding to the sequence shown in any one of SEQ ID NOs: 157 to 171, SEQ ID NO: 172 It is Sp2 corresponding to the sequence shown in any one of SEQ ID NOs: 185, 186 to 190, or Sp9 corresponding to the sequence shown in any one of SEQ ID NOS: 186 to 190, or equivalent to the sequence shown in any one of SEQ ID NOs: 191 to 204 Hump20, or Hump203 corresponding to the sequence shown in any one of SEQ ID NOS: 205 to 210.
[0010]
In another aspect of the present invention, there is provided a DNA having the sequence shown in any one of SEQ ID NOs: 8 to 238 or an orthologous DNA which hybridizes with the above DNA under stringent conditions.
The PCR primer can be selected from the group consisting of SEQ ID NOs: 276-325. The animal can be a mammal. Such mammals may belong to the artiodactyla order.
[0011]
In another aspect of the invention, there is provided a method of obtaining a consensus sequence of the SINE subfamily, comprising the following steps:
Sequencing multiple copies of the repeat unit by whole genome transcription in vitro or automated sequencing of genomic DNA larger than 60 kbp;
Aligning the above sequences from several species to obtain a consensus sequence;
Finding the consensus sequence of the second promoter for RNA polymerase III and creating a stem-loop structure containing this sequence;
Confirm that the sequence is 3'-PyPyPuPuPu-5 'with 5 bases 5' upstream from the stem region as one unit;
the next five bases forming the anticodon-stem region of the tRNA structure are considered other units;
The next seven bases forming the anticodon-loop region of the tRNA structure are considered as further units, where the 3 'end is an AA residue and the 5' end is 3'-TC-5 ' Confirm that it is a residue;
The next 5 bases were considered as other units and the formation of base pairs with the 5 bases assigned to the anticodon-stem region was confirmed, where 3 ′ of the anticodon-stem to align this unit correctly. Placing the deletion at the position of the first base on the side;
Constructing a stem-loop structure for the D region of this tRNA-like structure, confirming the presence of two Gs in the first promoter region of the RNA polymerase III;
Searching for sequence similarity between the sequence and the tRNA using a DNA database to confirm similarity in its secondary structure;
Arranging the SINE family sequences obtained as described above from a plurality of species to obtain the SINE subfamily from the presence of characteristic nucleotides or deletions;
Obtaining a consensus sequence of the SINE subfamily from the alignment of the SINE subfamily,
The method is provided.
[0012]
The SINE subfamily can be a DNA having a SINE subfamily consensus sequence obtained by the above-described method for obtaining a SINE subfamily consensus sequence.
In another aspect of the present invention, there is provided a DNA having a SINE subfamily consensus sequence obtained by the above-described method for obtaining a SINE subfamily consensus sequence.
[0013]
Definition
In the specification of the present application, “SINE” is a short scattered repetitive sequence having a primary structure length of about 100 to 400 bases, and a tRNA homologous region and a tRNA heterologous sequence in order from the 5 ′ end. Region, including those rich in AT. At both ends, there are characterized by direct repeats of about 5 to 20 bases which are considered to be formed when SINE is inserted into the genome.
As used herein, the “SINE family” refers to members of SINE that are derived from the same tRNA and have similar sequences.
On the other hand, in the present specification, the “SINE subfamily” is derived from the same origin (family), but has characteristic mutations. Therefore, the SINE subfamily was amplified from the same ancestral member in the SINE family. A member of a group. Subfamily is used interchangeably with type.
As used herein, an orthologous locus means that two loci are derived from speciation from a common ancestor. On the other hand, when two genes arise due to gene duplication rather than speciation, they are "paralogous". It is the orthologous gene, not the paralogous gene, that provides the information necessary for estimating the molecular phylogenetic tree.
[0014]
As used herein, the term "flanking sequence" refers to a sequence located on both sides of the insertion site when the SINE family or subfamily is specifically inserted into the orthologous locus. PCR primers can be arbitrarily designed within such flanking sequences.
In the present specification, the stringent condition means that a template DNA is put in a hybrid bag, a suitable amount of a prehybridization solution (6 × SSC, 1% SDS) is added thereto, the hybrid bag is packed with a sealer, and incubated for 1 hour or more. After discarding the solution and adding a hybridization solution (6 × SSC, 1% SDS, 1 × Denhart's solution, Carrier DNA (Shared Herring Sperm DNA solution)), the probe prepared in advance was heat denatured at 95 ° C. for 3 minutes. Then, the hybrid bag is sealed and the hybrid bag is sealed, and then incubated overnight (about 15 hours) in a water bath at 42 ° C., and the membranes are washed with an appropriate amount of a wash solution (2 × SSC, 1% SDS). Lightly Idatoki, probe refers to conditions that hybridize to the template DNA.
[0015]
In the present specification, the term “outer group comparison” refers to an outgroup, ie, a species or species group that is assumed to be most closely related to a target organism group, ie, an ingroup, in phylogenetic estimation theory. It refers to a method of determining trait polarity based on By including the outer group in the analysis, the location of the inner group root (common ancestor of the entire inner group phylogenetic tree) can be determined. In the early theory of bifurcation taxonomy, the polarity of the trait state of the inner group, that is, the primitive trait state and the derivative trait state, was first determined based on the trait state of the outer group, and then, it was determined in advance. Based on the trait polarity, species that share a derivative trait state are grouped together as a monophyletic group. Outer group comparisons are widely used, among other things, as the primary method of determining the polarity of morphological traits. The rationale for this is called the principle of maximum savings, in which the virtual trait state (criterion for polarity discrimination in the inner group) at the branch connected to the inner group root is estimated in the most conservative manner from the trait distribution of the outer group. Because there is. Maddison, Donogue & Maddison (1984) and Swofford & Maddison (1987) are the most conservative phylogenetic estimation of the inner group based on the comparison of the outer group without performing the polarity judgment for the group combining the inner group and the outer group. Has been proved to be logically equivalent to
Therefore, it has become possible to estimate the least conserved strain based on branching taxonomy even from molecular data for which polarity determination is difficult, such as restriction enzyme cleavage sites and nucleic acid base sequences.
[0016]
In the specification of the present application, the “shared derived trait” refers to sharing a trait state (apomorphy) determined to be derivative as a result of estimating the polarity using an outer group comparison or the like. The theory of clad taxonomy argues that only shared derivative traits provide information for estimating phylogenetic relationships. This is because a direct common ancestor that produced a group of species that share the estimated derived trait can be assumed. Therefore, the shared derived trait is a key to constructing a single lineage (more precisely, a complete lineage). Here, if there is an inconsistency in the trait distribution, the derivative trait state of some traits must be considered to be homoplasty that evolved on separate branches rather than from a common ancestor. The validity of the shared derivative trait is verified by its consistency with the phylogenetic hypothesis, which is selected based on the principle of least cost, that is, the clade diagram.
[0017]
As used herein, “polarity” refers to the direction of evolution of the transition order between the trait states of a certain trait. Therefore, the polarity depends on the model of the transition order. The transition order of the trait state reflects the trait of the trait data used for lineage estimation. It is not uncommon for qualitative morphological traits to assume a linear or branched transition order. On the other hand, many molecular data such as nucleic acid base sequences and restriction enzyme cleavage sites are unordered traits that cannot be ordered among trait states. Branching taxonomy and evolutionary taxonomy require polarity estimation of trait evolution prior to phylogenetic analysis. As a criterion for estimating the polarity, outer group comparison or information on fossil records or ontogeny is used, but the outer group comparison is most widely used.
[0018]
Evolution of SINE
It is important to understand how SINE has evolved in order to effectively utilize SINE in the lineage estimation, the isolation / identification method and the species identification method according to the present invention. SINEs can be divided into families based on sequence similarity and subfamilies based on characteristic nucleotide occurrences and / or deletions, as described in detail below. The fate of SINE, once inserted into the genome, is determined by the balance between various factors in the chromosomal environment (Schmit and Maria 1992) and mutations that prevent amplification that accumulates in SINE. . Furthermore, since LINE-encoded RTase is required for amplification of SINE, if LINE loses its ability to amplify in the genome and dies, it means that SINE also died in the organism. (Okada et al., 1997).
[0019]
Regarding how SINE amplifies in the genome during evolution, two alternative models of SINE evolution have been proposed so far: a master gene model and a multi-source gene model. The master gene model (FIG. 2A) shows that only one or several "master" loci are the source of all progeny copies, which have no ability to replicate on themselves. That is. In this scenario, its amplification over time is completely dependent on the condition and activity of the master gene. On the other hand, the multi-source gene model (Fig. 2 (B)) indicates that offspring can function as a "multi-source gene" over the course of evolution because they can have the same amplifying ability as the parent copy. is there. In the latter model, the amplification factor is a function of the rate of increase or decrease in total copy number from all of the source genes. The master gene model was proposed with empirical evidence from rodent ID SINE (Kim et al, 1994) and early studies of the human Alu repeat (Shen et al., 1991). However, due to subsequent detailed Alu sequence studies and comparative studies from various other taxa, it is now most pertinent to interpret that most SINE family amplifications occur with the multi-source gene model described above. (Matera et al, 1990; Schmid and Maraia, 1992; Leeflang et al, 1992; Kido et al., 1994; Takasaki et al. 1994; Shedlock and 2000). In practice, subfamilies characterized by the presence and / or deletion of characteristic nucleotides will represent each source gene.
[0020]
The period between the birth and death of SINE is the period of activity of SINE in the host genome, and its lifespan is directly related to its use in taxonomics and as a tool in the present invention. If the average sequence divergence between members of the SINE subfamily is small, it is reasonable to assume that the subfamily is quite young and is still actively amplifying in its host. If the average sequence difference between members of the SINE subfamily is large, it is reasonable to assume that the subfamily is relatively old and already inactive or dead in its host. However, diagnosis of common ancestry among host taxa using SINE insertion is only possible within the active life of a given subfamily. The basic principle of the SINE method will be further described in the “Procedure” section below.
[0021]
SINE insertion dynamics and feature theory
The key to why SINEs are powerful taxonomic tools is that they are independently and irreversibly inserted into the host genome (Murata et al, 1993; Shedlock and Okada 2000). Because there is no known mechanism to specifically remove SINE from the genome, and the probability that two elements are inserted or exactly excised from exactly the same locus is very low. One can regard the presence of SINE at the same locus in two different taxa as a polarized derivative phenotype in its genome. This defines one clade or monophyly in the strict sense of the method of Hennig (1966), which uses only shared derivative traits, or shared derived traits, to construct the phylogenetic hypothesis. In this clade, known ancestral conditions, or the lack of a SINE insertion at a given locus, uniquely define the outer group without the need to establish characteristic polarity via competitive methods of creating known artifacts. (Hendy and Penny, 1989) (see FIG. 3).
[0022]
Methods for isolating and characterizing SINE
Hereafter, strategies for isolating novel SINEs from genomic libraries, screening methods thereof, sequencing of clones and characterization of SINE families from subfamilies, quantification of copy number in representative host taxa, and The definitive diagnosis of the SINE insertion pattern providing phylogenetic information is described.
[0023]
procedure
How to select species to create a genomic library
The SINE method comprises the following basic steps: 1) creation of a genomic library from a selected species; 2) isolation of a clone containing the SINE locus; 3) determination of the DNA sequence of the clone; 4) its SINE gene. Design of polymerase chain reaction (PCR) primers within the flanking sequence of the locus; and 5) PCR diagnosis of the presence or absence of SINE between related species of interest. Although the SINE method can provide deterministic results quickly and reliably once established, it requires considerable time and effort to establish and establish its procedures and conditions.
[0024]
For example, consider the case of determining a phylogenetic relationship between closely related species A, B, C, and D. The actual phylogenetic tree of the above species is shown in FIG. In this case, if species D is selected as a host from which to isolate SINE, no SINE locus providing phylogenetic information is obtained. Species D contains the SINE locus inserted within the common ancestry of the four taxa of interest and within older sources. The locus inserted into all four ancestors is shown as SINE3 in FIG. 4 (B). The pattern of the electrophoresis gel of PCR showing the presence (+) or absence (-) of SINE3 insertion is shown in the lower part of FIG. To isolate a SINE that provides phylogenetic information, one must select species A or B that can provide the three SINE loci, SINE1, SINE2, and SINE3, shown in FIG. 4 (B). Must. Each of these loci defines a clade, or monophy, that shares a common ancestor in the evolutionary history of these species.
[0025]
Method for isolation of novel SINE family from specific species
When no SINE family is known for a particular species, eg, species A in FIG. 4 (A), it is necessary to newly isolate and analyze the SINE family from its genome. There are two ways in which a new SINE family can be isolated from a selected species. One involves in vitro whole genomic DNA transcription (Endoh and Okada, 1986), and the other involves genomic DNA sequencing of about 60 Kbp or more (Nikaido and Okada) facilitated by a new high-throughput automated DNA sequencing method. , Not announced).
[0026]
In vitro whole genomic DNA transcription
Most SINEs are known to be derived from tRNA; therefore, SINEs have an internal promoter for RNA polymerase III. Although SINE is very rarely transcribed in vivo, SINE can be easily transcribed in vitro from naked DNA. When transcribing certain total genomic DNA in HeLa cell extracts using a radiolabeled precursor nucleotide, eg, alpha P32-GTP, radiolabeled RNA is usually transcribed. In some cases, these radiolabeled transcripts form a clear band when they are subjected to gel electrophoresis (Endoh and Okada. 1986; Matsumoto et al. 1986).
[0027]
This radiolabeled RNA can be used as a probe to screen a genomic library from the selected species of interest. If this transcript forms a clear band in the gel, it represents the actual SINE family. This is because all of the same transcripts from each given locus of the SINE family collectively form distinct bands. Even when obscured bands arise from genomic DNA, they can be used as probes for screening. However, in the latter case, the transcript may represent multiple SINE families.
[0028]
Our experience shows that when vertebrate and / or invertebrate genomes have more than 10,000 copies of SINE, they are detected by in vitro transcripts of whole genomic DNA. Can be. FIG. 4 shows some example patterns of transcripts from total genomic DNA from selected animal species (Endoh and Okada, 1986).
[0029]
Sequencing of genomic DNA larger than 60 kbp by automated sequencer
According to the experience of the present inventor, the copy number of the SINE family in the genome is usually over 10,000. The whole genome is 3x10 in length⁹bp and the size of a SINE is 300 bp, such a SINE family would occupy 0.1% of its genome (eg, 300 x 10⁴= 3x10⁶). Thus, by sequencing over 60 kbp, one would be able to find two independent SINE sequences within this type of randomly isolated DNA fragment (eg, 600 × 100 / 0.1 = 6 × 10⁶).
[0030]
This has become easily achievable in the laboratory with access to the latest models of high-throughput automated DNA sequencers. For example, a new SINE family has recently been characterized from the genome of elephants, and it has been shown that this new SINE is distributed among all species of Afrotheria (Nikaido and Okada, unpublished). This method can be applied to the genomes of all mammals and possibly most vertebrates.
[0031]
Method for accurately identifying the SINE family and deducing its tRNA structure
After determining the sequence of multiple copies of the repeat unit according to the methods described above, they can be aligned and a consensus sequence for the repeat family can be deduced. Since there are many repetitive sequences in the genome other than the SINE element, it is essential to appropriately diagnose the sequences. Since most SINEs are known to be derived from tRNA, they contain a promoter for RNA polymerase III. The RNA polymerase III promoter is a conserved sequence block that has the properties of a first promoter and a second promoter separated from one another in its genome. This second promoter is highly conserved and can be easily recognized empirically.
[0032]
Hereinafter, an example of CHR-2 SINE will be considered. The tRNA-like structures of these elements can be established as follows (see FIG. 6):
1. First, a CHR-2 SINE consensus sequence is constructed from the alignment of several CHR-2 sequences (see FIG. 8).
2. Visually search the second promoter consensus sequence for RNA polymerase III. This sequence is 5'-GT (or A) TCG (or A) -3 '. There are no exceptions to this motif when screening this promoter. When this motif is present, it creates a stem-loop structure containing this second promoter sequence. The number of bases in the loop is 7 and the number of base pairs in the stem is 5. Even if all of the bases in the stem region do not form base pairs, place those bases at appropriate positions in the tRNA, as shown in FIG. 6 (A).
[0033]
3. One unit is assumed to be 5 bases 5 ′ upstream from the stem region. This is because in the plasma class ItRNA, this extra loop region consists of 5 bases. In the case of CHR-2 SINE, the sequence of this unit of 5 bases is 3'-CAGGG-5 '. The 3'-PyPyPuPuPu-5 'sequence is typical of this extra loop in some tRNAs (Sprinzl et al. 1987) and this allows deduction of the tRNA origin of the SINE (FIG. 6 ( B)).
4. The next 5 bases forming the aminoacyl-stem region of the tRNA structure are considered as other units. In this case, it is 3'-GACGT-5 '(see FIG. 6C).
5. The next seven bases forming the anticodon-loop region of the tRNA structure are considered as further units. In this case, it is 3'-AACCGTC-5 '. AA residues at the 3 'end 3'-TC-5' residues at the 5 'end are good indicators of the tRNA origin of this SINE. This is because these bases are highly conserved in most tRNAs (Galli et al., 1981). This further supports the tRNA origin of this SINE (see FIG. 6 (D)).
6. Normally, the next five bases are considered other units, and they should base pair with the five bases assigned to the anticodon-stem region (FIG. 6 (C)). In this case, the sequence is 3'-CGTCT-5 ', and only its first four bases match well with the anticodon-stem region partner. In order to correctly align this unit, a deletion is placed at the position of the first base 3 'to the anticodon-stem (see FIG. 6 (E)).
7. Next, a stem-loop structure for the D region of this tRNA-like structure is constructed. The number of bases in this stem and loop is usually 4 and 8, respectively, but can vary by about 1 to 2 bases, especially in the tRNA-like structure of SINE. Clearly, some base pairs next to the CHR-2 do not form a meaningful secondary structure. In this case, attention is paid to the first promoter region. The most striking feature in the first promoter region is the presence of two Gs in that region. Other features are G in position 15 in this loop (depending on the tRNA numbering system; the same applies hereinafter) and A in position 14. A at position 14 is the first base in the loop on the 5 'side. Therefore, these bases are arranged at the corresponding positions of the loop of this tRNA-like structure (see FIG. 6 (F)). It is well known that T, the first base in FIG. 6 (F), is highly conserved in all tRNA molecules.
8. The tRNA-like structure of CHR-2 SINE can be deduced by combining the sequence shown in FIG. 6 (F) with other sequences for this family (FIG. 7 (A)).
9. Next, the BLASTN program in the GenBank DNA database is used (Altschul et al. 1990) to search for similarities between CHR-2 SINE and the actual tRNA. In this example, tRNA Glu is most similar to the sequence of CHR-2. FIG. 7 (B) shows the secondary structure of human tRNA Glu.
[0034]
How to characterize the SINE family into subfamilies
Suppose that a SINE family is characterized in the genome of species A in phylogeny, and it is unknown when this SINE family was first generated during evolution. Further assume that this SINE family was first generated in the old common ancestor of all taxa of clade Y in FIG. In this case, the copies of SINE present in the genome of species A include the old SINE amplified at t in FIG. 9 and the young SINE amplified at u. When screening a species A genomic library with this SINE family consensus sequence, both the old and new SINEs described above can be isolated. Since only the phylogenetic relationships of species A, B, C, and D are sought, it is inefficient to examine all times of an isolated SINE amplification event. Rather, it is much more efficient to attempt to isolate the SINE locus that is amplified near the divergence of species D and spans all branches of the four taxa, including clade X in FIG.
[0035]
As described above, SINE and LINE are believed to be amplified according to a multi-source gene model (Schmit and Maria 1992; Smit et al. 1995). If a source gene is mutated and successfully amplified during evolution, the mutated source gene can be recognized as a subfamily within its corresponding SINE family (Britten et al. 1988). Jurka et al., 1995). Subfamilies are amplified at some stage in evolution. Therefore, if a subfamily is amplified only in the common ancestor of species A, B, C, and D, copies of this subfamily can be used effectively to determine the phylogenetic relationship of these four clades Can be done.
[0036]
The SINE family consensus sequence in species A was established as part of the above procedure and allowed the design of one PCR primer at the 5 'end of the SINE sequence and other primers in a conserved region near its 3' end. I do. The sequence amplified by this primer set encompasses the entire SINE sequence. This primer set can be used to amplify many copies of SINE by PCR using genomic DNA from species A. The PCR product of this reaction can be cloned into a suitable vector DNA and sequenced. At this point, sequencing 100 copies of SINE from the PCR product is not a difficult task. By aligning these sequences, characteristic nucleotides or possible deletions representing this SINE family subfamily can be identified (see FIG. 10). Characteristic nucleotides are defined as those that are cooperatively altered at three or more nucleotide positions within a particular subfamily and that can be distinguished from spontaneous mutations that have accumulated at random within the SINE sequence during evolution. Is done. Only after successfully characterizing a subfamily based on the presence of characteristic nucleotides and often specific deletions, the taxonomic distribution of a given subfamily can be determined by dot-blot hybridization or PCR. Become like
[0037]
CHR-2 SINE subfamily
FIG. 8 shows the CHR-2 SINE family (Shimamura et al. 1997) originally characterized as being present in the genomes of the orders Cetaceans, Hippopotamus, and Ruminants. Shows copy alignment. It is readily apparent that CHR-2 SINE has six subfamilies. FL (full length), MDI (central deletion I), MDII (central deletion II), and shortest groups were characterized due to the presence of the deletion. This shortest group can then be classified into the subfamilies DT (deletion type), CD (whale deletion type), and CDO (whale deletion tooth specific whale). FIG. 11 shows the alignment results of the consensus sequences of these subfamilies. SEQ ID NO: 1 is the upper consensus sequence, SEQ ID NO: 2 is the FL consensus sequence, SEQ ID NO: 3 is the MDI consensus sequence, SEQ ID NO: 4 is the MDII consensus sequence, SEQ ID NO: 5DT consensus sequence, SEQ ID NO: 6 is the CD consensus sequence, and SEQ ID NO: 7 represents the CDO consensus sequence.
[0038]
FIG. 12 shows the results of dot-hybridization experiments using probes specific to CD, CDO, and other subfamilies, respectively. This result clearly shows that the CD subfamily is specific for the genome of the order Whale (Dental and Beard Whales), and that the CDO subfamily is specific for the genome of the tooth whale. Therefore, SINE belonging to the CD subfamily is useful for estimating the phylogenetic relationship of whales, especially bearded whales, while SINE belonging to the CDO subfamily is useful for estimating the phylogenetic relationship of tooth whales. Useful. The distribution of copies of the CDO subfamily also suggests monophyly of the subclinical whales, including sperm whales, which was one of the most controversial points in the taxonomic biology of mammals.
[0039]
Flanking SINE PCR
After isolating the SINE loci from species A and belonging to the subfamily generated in the common ancestor of clade X shown in FIG. 9 and determining their sequence, the presence of an insertion at a given locus or PCR experiments can be performed to diagnose the absence. Looking at the flanking (adjacent) sequence, a primer sequence is selected. Care must be taken in designing primers for the formation of secondary structure folds and for tandem annealing between the upstream and downstream primers. This can be easily checked using a variety of standard software programs written to facilitate PCR primer design, commercially or via the Internet. The melting temperature of the oligonucleotide primer is set around 55 ° C. Therefore, the annealing temperature for PCR must be based on this temperature when optimizing the amplification of orthologous loci from species B, C, and D. Sometimes the accumulation of mutations in the primer binding region for species B, C, and D prevents efficient primer-template annealing during this reaction. In this case, the annealing temperature must be reduced to about 45-50 ° C. If the PCR product is not amplified by PCR with the first primer, new PCR primers should be designed with further care for potential artifacts that may reduce the efficiency of the PCR. FIG. 13 schematically shows the principle of flanking SINE PCR.
[0040]
FIG. 14 shows an example of the PCR result. Also, the results of hybridization experiments performed using the same filter using two different probes are shown simultaneously. FIG. 14 (A) is a PCR pattern that provides evidence that sea dolphins are a single line. This is because PCR products from marine dolphins have the expected fragment size containing the inserted elements, while fragments from other tooth whales have the expected fragment size lacking the insertion in Mago 19. FIG. 14 (B) shows a hybridization experiment using the SINE probe, while FIG. 14 (C) shows a hybridization experiment using the flanking DNA at the above locus. This latter experiment was performed to demonstrate that the orthologous loci were faithfully amplified by PCR in species other than Magondo from which the loci were originally isolated and characterized.
[0041]
Interpretation of PCR data
When examining relatively recently diverged species, the flanking sequence at the orthologous SINE locus is faithfully conserved, and typically does not cause problems that inhibit PCR diagnosis. However, where relatively old divergent taxa are examined, PCR fails more frequently and makes interpretation of experimental results difficult. In an unsuccessful PCR, SINE-minus data appears. That is, there is a successful PCR amplification at a given locus indicating the absence of a SINE insertion. Unsuccessful PCRs represent lost data and are coded as is (eg, "?") When performing the most parsimonious analysis of the SINE character matrix encoding patterns with or without insertions.
When there is a conflicting insertion pattern between the examined independent loci, there should be an ancestral polymorphism and subsequent incomplete phylogeny.
[0042]
Distribution of SINE in mammalian genome
Generally, most mammals have a large amount of SINE. They are apparently specific to the order, suborder, superfamily, family, genus, or species, based on the hybridization pattern between the species examined (eg, the distribution of CHR-2 SINE in FIG. 12). . Such empirical evidence indicates that the SINE family is newly generated in many ancestral mammalian strains. However, its generation mechanism is not fully understood. The reason for such a large number of SINEs or retroposons in the mammalian genome may be due to the fact that the RTase encoded by the mammalian L1 has altered its template recognition specificity in a common mammalian ancestor. . This allows the recognition of the poly A tail required for retropositions present in many LINEs that strictly recognize the 3 'tail responsible for the formation of the stem-loop structure (Ohshima et al. 1996; (Okada et al. 1997; Kajikawa et al.). Such a scenario could have enabled poly-A-containing RNA to become a pseudogene via L1 RTase in the mammalian genome.
[0043]
FIG. 15 shows a recently proposed mammalian phylogenetic tree (Waddell et al. 1999; Cao et al. 2000; Nikaido et al. 2000). The mammalian SINE family that has been characterized to date is described on FIG. Briefly, the oldest SINE family distributed in the entire mammalian genome is the MIR (Smit and Riggs 1995; Jurka et al. 1995). Although the Alu family is clearly specific to the primate genome, its distribution among closely related species, such as lemurs, has not been investigated in detail. Rodents B1, B2, and ID are specific to the rodent genome. The rabbit C family has been reported in the genome of Lagomorpha, but its distribution among closely related species has not been reported. The SINE family present in the whale artiodactyl genome, for example, CHR-1, CHR-2, CHRS, CHRS-S, PRE-1, and Bov-tA, has been investigated in detail (Shimamura et al. 1997; Shimamura et al. 1999; Nikaido et al. 1999). Although CanSINEA was first reported from the Canidae genome (Minnick et al. 1992; Coltman and Wright 1994), it was subsequently shown that it occurred during evolution within many other carnivorous genomes (van der Vlugt and Lenstra 1995). The equine SINE, named the ERE family, has been reported and its distribution examined (Sakagami et al. 1994; Gallagher et al. 1999). The bat SINE family was isolated by Borodulina and Kramerov (1999) and named VES, and other bat SINE families have recently been characterized (Kawai et al.). The elephant SINE family has recently been isolated and shown to be distributed among the species of Afroteria (Nikaido and Okada). Here, SINEs having different or totally different tRNAs are distinguished as family, and SINEs derived from the same source but having a diagnostic mutation are identified as Type or subfamily. It is considered that a certain SINE explosively increases its copy number at a certain time, and its explosive amplification time and the time axis in the evolution of the whole organism are linked to the relationship between their phylogenetic relationship and the distribution of the SINE.
[0044]
Importantly, although many SINE families have been isolated to date in the mammalian genome, they have not yet been characterized to their subfamily structure. Thus, in accordance with the present invention, its subfamily structure will be elucidated in the future, and new mammalian SINE families or subfamilies may be further obtained.
[0045]
New amplification of SINE: fixed and short-term species divergence
If SINE is very newly amplified in genome evolution and is not fixed among a population of one species, its status as a common derivative is unstable and it should be used for phylogenetic tree construction is not. However, such a distribution of SINEs can be used for analysis of population structure (eg, Hamada et al. 1998). If species divergence occurs in a short period of time, ie, before most SINEs are fixed among populations via genetic drift, ancestral polymorphisms and subsequent incomplete phylogeny will result in inconsistent SINEs. Create an insertion pattern. This phenomenon, combined with the irreversible nature of the SINE retroposition, provides a bias for using SINE to examine the historical pattern of phylogeny in explosively derived taxa. If there is a polymorphism in the ancestor, and then the SINE is incompletely distributed to the offspring, creating an inconsistent insertion pattern, one of the phylogenetic tree construction methods, the most conservative method, can be used at each locus It is useful to evaluate the SINE character matrix for the presence or absence of the insertion. Unfixed, polymorphic SINEs are not useful for high-level phylogenetic relationships, but they are known to be excellent taxonomic tools for population analysis (Deininger and Batzer, 1994, Stoneking et al, 1997; Hamada et al, 1998). Therefore, by identifying and characterizing the SINE family or subfamily according to the method described in the present specification, an animal species discrimination method can be performed using the SINE family or subfamily.
[0046]
Function and value of flanking sequences (usefulness)
Information of nucleotide flanking sequences within the locus examined for SINE insertions is useful. Of course, although the insertion data is useful for generating a phylogenetic tree and for use in the method of the present invention, the integration of the insertion sequence with the flanking sequence is dependent on the length of the branches between the clades determined by the independent SINE insertion case. To provide information about the protein (Lum et al, 2000; Shedlock and Okada, 2000). In addition, since the flanking sequences associated with a given insertion element are literally linked, the consistency between the SINE-derived topology versus the flanking sequences may be a factor in assessing the basic assumption of irreversible insertion at each locus. It offers a number of approaches (Lum et al, 2000). If there is a homopathy (genetic homology) or trait discrepancy at independent SINE loci, there may be a clear discrepancy between the phylogenetic trees. Such an approach is starting as a new dimension of SINE analysis and provides the basis for enhancing the statistical evaluation of the SINE method.
[0047]
【Example】
Hereinafter, the present invention will be described in detail with reference to examples. However, the technical scope of the present invention is not limited by the following examples.
[0048]
Materials and methods
Buffers, enzymes and other reagents
Various buffers, enzymes, culture media for culturing Escherichia coli, and other reagents used for nucleic acid manipulation were purchased from Wako Pure Chemical Industries, Ltd., Sigma, Difco, and FMC, and purchased from Sambrook et al. (1989). Various restriction enzymes, modification enzymes, and vector DNA were purchased from Takara Shuzo Co., Ltd., Toyobo Co., Ltd., and Amersham Life Science Company. Radioisotopes were purchased from Daiichi Pure Chemicals. The PCR primers used were those ordered by OligoExpress (Amersham Pharmacia Biotech) for synthesis.
[0049]
genome DNA
Various samples (tissue or DNA) used in this study are shown below. Table 1 below shows the English names, Japanese names, scientific names, etc. of whales and artiodactyls used in the analysis.
[Table 1]

Shown in
Bactrian camel extracted DNA from muscle tissue provided by Dr. Minoru Yoshida of the University of Tokyo School of Medicine. For pigs, the DNA stored in this laboratory was used as it was. Peccary used DNA provided by Dr. Hiroshi Yasue of the Ministry of Agriculture and Livestock Experiment Station. Java deer used DNA provided from the San Diego Zoo in the United States. For Axis deer, Amimekirin, Sable antelope, Japanese serow, Markor, Mflon, and Hippo, DNA provided by Mr. Isao Munechika of Chiba Zoological Park was used. The sheep used were extracted from the liver provided by Yasushi Mukai of the Ueda Meat Sanitation Laboratory in Nagano Prefecture. For cattle, DNA was extracted from the kidney provided by the Kanagawa Prefectural Meat Sanitation Laboratory Sagami Branch and used.
[0050]
As for the samples of narwhals and giant whales, DNAs were extracted from DNA fragments extracted from a tissue piece considered to be muscle from Dr. Masaki Miya of Chiba Prefectural Central Museum and used. It is known that the sample of the giant whale is belonging to the genus Giant Sperm, but the species name is unknown. Other samples except for the dolphins were used by extracting DNA from various tissue pieces provided by Dr. Hidehiro Kato of the Large Whale Laboratory of the Fisheries Agency, Ocean Fisheries Research Institute. For samples of dolphins, see Dr. Robert L. Brownell, Jr .: Chief Marine Mammal Division, Southeast Fisheries, California, California, USA, USA, Dr. Robert L. Brownell, Jr., 27th. We had you perform acquisition procedure to Japan. Based on that, a subcutaneous tissue section of Amazon dolphin and a liver tissue section of Laplata dolphin were described by Heally H. of the University of California, Berkeley. DNA was extracted from Hamilton. Ganges dolphins provided DNA from dried bone samples provided by Tadashi Yamada of the National Museum of Nature and Science. As for the dolphins, the freshwater was collected from an individual kept in the aquarium of the Institute of Aquatic Biology of the Chinese Academy of Sciences, treated with heparin on the spot, transported to the laboratory, and immediately extracted with DNA. Since the extracted DNA cannot be brought into Japan, it is currently stored at a Chinese laboratory.
[0051]
genome DNA Extraction
Genomic DNA was extracted from the tissue of the animal species used for the analysis by a simple method (Rapid method; a method using Proteinase K). The extracted genomic DNA was used as a template for library preparation and PCR. They are also stored at 4 ° C. Tissues (liver, muscle) of whales and cloven-hoofs stored in a freezer at −40 ° C. are cut out into several millimeter squares with a scalpel, transferred to a microtube, weighed, and then 1X into the microtube. 500 μl of TNE buffer (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0)) was added. When the tissue piece in the tube is finely pulverized using a Pasteur pipette, the tissue and the TNE buffer are subjected to 1X Lysis buffer (20 mg / ml Proteinase K, 2% SDS, 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM). EDTA (pH 8.0)) was added, and the mixture was stirred by inversion, and then incubated using a 55 ° C water bath to completely dissolve the tissue pieces. An equivalent amount of phenol was added to the solution, and the mixture was stirred with a belly dancer (200 to 300 rpm) for 1 to 2 hours. After centrifugation (room temperature, 12000 rpm, 5 min), the supernatant was transferred to another 2.0 ml microtube. In the same manner, phenol / chloroform extraction and chloroform extraction were repeated, and 0.1 times volume of 3M sodium acetate and 2 times volume of ethanol were added to the final supernatant. At this time, in many cases, a fiber was confirmed. Thereafter, the fiber was rinsed with 70% ethanol, and then an appropriate amount of TE buffer was added to dissolve the pellet. Extraction of DNA from a blood sample of Aedes aegypti was performed using a commercially available column (QIAamp Tissue / Blood Kit Cat. No. 29306: manufactured by Qiagen Co., Ltd.).
[0052]
Flanking PCR (Mainly used for amplification of homologous loci)
Flanking PCR means that primers are designed on both sides (upstream and downstream) of the SINE sequence so as to sandwich the SINE sequence, and PCR is performed using genomic DNA as a template. The primer used at that time was designed to have a length of about 17 to 30 nucleotides and a Tm (melting temperature) value of about 55 ° C. With regard to the design, a CP Primer (Ver. 1.08, Bristol and Anderson (1995)) which can be downloaded on the Internet as Macintosh version free software was used.
Reaction composition: Template DNA (100 to 500 ng); 10X PCR Buffer (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl)₂  5 μl; dNTP Mixture (2.5 mM each) 4 μl; Forward and Reverse Primer (5 pmol / μl) 2 μl each; TaKaRa TaqTM (5 U / μl) 0.25 μl; addsd₂50 μl of O to to final volume.
Reaction conditions: 94 ° C. (Pre-denature) 2-3 min. And 30 cycles of 94 ° C. (Denature) 30 sec. 45 ° C to 60 ° C (Annealing) 1 min; 72 ° C (Extension) 30 to 90 sec. . At this time, the optimum annealing temperature was determined by appropriately changing the annealing temperature according to not only the Tm value of the primer but also the easiness of increasing the number of loci.
[0053]
colony PCR
Colony PCR is a very simple method for selecting whether or not a target DNA fragment is contained in a certain clone as an insert when performing subcloning. Conventionally, plasmid DNA was prepared by Miniprep and then digested with a restriction enzyme to confirm the presence or absence of the insert. However, since this step can be performed only by PCR, a significant amount of time and labor is required. It leads to shortening. First, colonies of Escherichia coli grown on the plate are gently plucked with a toothpick, rubbed against a 0.5 ml PCR microtube, and then used as a template to perform a normal PCR in a 20 μl reaction system. The primer used at this time is prepared based on a sequence specific to the vector. During thermal denaturation in the PCR reaction, the cell membrane of Escherichia coli is destroyed, and the vector DNA serving as a template dissolves into the solution, thereby enabling this PCR. As will be described later, after confirming the presence or absence of the insert by this PCR, this PCR product can be directly used as a template for sequencing using an ABI sequencer, so that the insert can be sequenced immediately after the insert check. Was also considerably shortened.
[0054]
Determination of nucleotide sequence
For the determination of the nucleotide sequence, the following sequencer and sequence reaction kit were used. Sequence using LI-COR dNA Sequencer (Model 4000): SequiTherm EXCELL (TM) II Long-ReadTM DNA Sequence Kit-LC (Cat. No. SE7701LC; Sequence used: BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (P / N 4303152, manufactured by Perkin Elmer).
When the subcloned plasmid DNA was used as a template for a sequencing reaction, a DNA prepared using SDS-Alkaline and Mg precipitation was used.
When the nucleotide sequence of the locus containing the SINE sequence isolated by screening is determined, M4 and RV on the vector sequence, and an oligonucleotide primer to which fluorescence has been prepared based on the SINE consensus sequence (Aloca ( Co., Ltd.) was used.
When the PCR product is directly sequenced, shrimp alkaline phosphatase (SAP) 2 U / μl 0.5 μl; Exonuclease I 10 U / μl 0.5 μl (both manufactured by Amersham Life Science) are directly added to the PCR product, and the mixture is added at 30 ° C. at 30 ° C. After decomposing the oligonucleotide by incubating for minutes, the enzyme inactivated by incubating at 85 ° C. for 15 minutes was used as a template for the sequencing reaction.
[0055]
Preparation of genomic library
Sucrose gradient
About 50 μg of genomic DNA was completely digested with an appropriate restriction enzyme (mainly Hind III was used in this study), extracted with phenol / chloroform, and precipitated with ethanol, and then dissolved in 200 μl of TE buffer. A 10-40% sucrose gradient was prepared in a 15 ml plastic tube for ultracentrifugation (Centrifuge Tubes-50 Ultra-Clear (registered trademark) Tube 14 X 89 mm, Order No. 344059: manufactured by BECKMAN), and DNA was placed thereon. The solution was added, attached to a rotor, and centrifuged (25000 rpm, 15 ° C., 15 hours) with an ultracentrifuge (L8-70M, Serial No. 7C869: manufactured by BECKMAN). After centrifugation, 10 to 15 drops were added dropwise to a 1.5 ml microtube to fractionate (about 250 to 400 μl / fraction). The fraction was subjected to 0.7-1% agarose gel electrophoresis, and 4-6 fraction tubes were selected so as to sandwich a fraction containing a fragment of about 2-4 kbp, followed by ethanol precipitation. Dissolved in TE buffer. 0.7-1% agarose electrophoresis was performed again, and the fraction used for library preparation was determined.
[0056]
Construction of plasmid library
In this experiment, only a plasmid library using pUC18 / HindIII or pUC19 / HindIII was used, and no phage library was prepared. Our laboratory knows that a sufficient number of SINE copies exist in the genomes of whales and artiodactyls, and that a sufficient number of positive clones can be obtained without using a phage library. In order to save time, a plasmid library with relatively few operation steps was used. The plasmid library was prepared as follows. The composition was TaKaRa Ligation Kit Ver. Solution A, 12 μl; Solution B, 1.5 μl; Vector DNA (pUC18 or 19 / HindIII to 100 ng / μl), 0.5 μl; Insert DNA (Genomic DNA Sucrose Radiation Fraction, 1.0 fraction) μl: After mixing the above solution in a 0.5 ml tube, the mixture was left on a cool block at 16 ° C. for 30 minutes or more. This genomic library is stored at -20 ° C.
[0057]
SINE Flow to clone isolation including sequence
FIG. 16 shows the flow of the experiment in the SINE method.
screening
Preparation of membrane used for screening
To a 0.5 ml tube, 1 μl of the above-mentioned genomic library mixture, an appropriate amount of competent cells of Escherichia coli (E. coli strain JM105) were added and mixed, and the mixture was allowed to stand on ice for 30 minutes. After heat shock at 42 to 45 ° C for 45 seconds, the plate was plated on an L / amp / X-gal / IPTG plate that had been incubated at 37 ° C in advance, and left overnight in a 37 ° C incubator. The number of colonies per plate was adjusted to 200 to 300, and a library for 10 to 20 plates was spread. The colonies that grew on this plate were transferred to a nylon membrane (Colony / Plaque Screen (trademark) NEF-978: manufactured by NEN Research Products), and denaturation solution (0.4 M NaOH, 0.6 M NaCl), neutralized The solution (1 M NaCl, 0.5 M Tris-HCl (pH 7.0)) was left in this order for about 3 minutes. The membrane was dried well after draining. After transferring the colonies, the plate was incubated at 37 ° C. so that the colonies were sufficiently grown again, so that the positive clones described later could be easily picked up.
[0058]
Preparation of probes used for screening
Oligonucleotides or PCR products were used for screening. The oligonucleotide sequences actually used are shown in Table 2 below:
[Table 2]

Shown in First, in the case of an oligonucleotide: 27 to 37 μl of ddH2O; 1 μl of Oligo Nucleotide (5 pmol / μl); 5 μl of 10 × T4 Polynucleotide Kinase Buffer 5 μl; Was mixed in a 0.5 ml tube and incubated at 37 ° C. The sequence of the oligonucleotide used as the probe is shown in Table 2 above. When a PCR product is used (Primer-Extension method): Template DNA, 500 μg; Forward primer (12.5 nmol / μl) 2 μl; Reverse primer (12.5 nmol / μl) 2 μl dDTP Mixture, GTP, GTP dTTP) 2.5 μl; The above mixture was heat-denatured at 95 ° C. for 5 minutes, then annealed at 55 ° C. for 1 minute, and then transferred to ice. Further, after mixing the following reagents in order, the mixture was incubated at 60 ° C. for 30 minutes. 2.5 μl of 10 × BcaBEST ™ Buffer; 2 μl of BcaBEST ™ DNA polymerase; [α- 32 P] dCTP. Each probe after the reaction was purified by elution with NICK Column (Sephadex G-50 DNA Grade: Amersham Pharmacia Biotech Co., Ltd.). The RI count was measured with a liquid scintillation counter. (A Geiger counter was also used for simple measurements.)
[0059]
Hybridization
The membrane was placed in a hybrid bag, and an appropriate amount of a prehybridization solution (6 × SSC, 1% SDS) was added thereto. The hybrid back was packed with a sealer and incubated for 1 hour or more. After discarding the solution and adding a hybridization solution (6 × SSC, 1% SDS, 1 × Denhart's solution, Carrier DNA (Shared Herring Sperm DNA solution)), the probe prepared in advance is heat-denatured at 95 ° C. for 3 minutes. Then, the hybrid bag was sealed, and the hybrid bag was sealed, followed by incubation overnight (about 15 hours) in a water bath at 42 ° C. The membrane was washed with an appropriate amount of a wash solution (2 × SSC, 1% SDS). After measuring and confirming the rinse count lightly, washing was performed by further setting the temperature of the water bath at 55 to 60 ° C. using a fresh washing solution.
[0060]
developing
After discarding the wash solution and rinsing lightly with a fresh wash solution, the count was checked using a Geiger counter. In a dark room, a cassette, a membrane, an X-ray film, and an intensifier were placed in this order, and the cassette was placed in a freezer at -80 ° C and exposed. The X-ray film was taken out of the cassette in the dark room, and the X-ray film was put into the developer, the stop solution, and the fixer in this order, and developed.
[0061]
Positive Clone Isolation
The position of the positive clone was confirmed by overlapping the developed X-ray film and the plate with the library. Before performing Miniprep on the colonies considered to be positive clones, primers prepared based on the consensus sequence of the SINE sequence used for screening instead of the vector sequence to confirm the presence or absence of the SINE sequence in the clone Was used to perform colony PCR. Thereafter, a plasmid of a positive clone was prepared, and the region near the plasmid was determined using an LI-COR sequencer. Since the subsequent operation is as described above, it will be briefly described below.
[0062]
First, primers were prepared based on the flanking sequences, and flanking PCR was performed using genomic DNAs of various organisms as templates. And, in many cases, homologous loci can be easily amplified for closely related species of the species used for screening, but only for homologous species that are likely to have a distantly related group or divergence in an earlier age. At the locus, primers based on one type of sequence used for screening did not anneal well because amplification of more base substitutions in the vicinity of the locus, and it was often observed that amplification was not possible with the first flanking PCR. Was done. At that time, the base sequences of the amplified homologous loci of various species were determined, a consensus was obtained, new flanking primers were prepared again in consideration of the consensus, and PCR was performed again.
[0063]
When estimating the phylogenetic relationship between whales and hoofed fishes, Southern hybridization was performed after agarose gel electrophoresis to confirm whether the amplified band was a homologous locus. The process is described below. After agarose gel electrophoresis, staining with ethidium bromide (EtBr) solution was performed, and the migration pattern was confirmed and photographed with a digital camera. Thereafter, one membrane and two filter papers sufficiently immersed in a denaturing solution (0.4 NaOH, 0.6 M NaCl) are placed on the gel in this order so as to prevent air bubbles from entering between them, and a JK wiper, Kim towel ( Trademark) and a dictionary as a weight, and left overnight (~ 15 hours). The membrane was neutralized by leaving it in a neutralization solution (1 M NaCl, 0.5 M Tris-HCl (pH 7.0)) for about 15 minutes, and then dried sufficiently. Subsequent operations such as hybridization and development are the same as those in the above-described screening, and thus description thereof is omitted. In addition, when estimating the lineage relationship in the order of whales, since their base sequences were determined for most loci, they were not basically confirmed by Southern hybridization.
[0064]
Example 1: Within the whale artiodactyl genome CHR-2 Distribution of each subfamily
Regarding the origin of whales, their internal phylogeny has been categorized by morphological classification and molecular classification. There are three subspecies of the order Coleoptera, which includes many indigenous species, the Coleoptera and the Beards, and the Genius Coleoptera (also known as the Whales), which is believed to have been the ancestor of these species. It is divided into eyes (Fordyce et al., 1994), and the classification of modern whales is distinguished by having or not having teeth, as is clear from the classification name. However, bearded whales have teeth that can be confirmed to the very last stage of the outbreak, and among the fossil species of original whales, naturally, traits that are thought to be in the middle stage between tooth whales and bearded whales There are also some with whales (that is, bearded whales with teeth), so as a matter of fact, it is not possible to assert the monophylicity of tooth whales simply by the presence of teeth. This is because, when considered systematically, the "whiskers" of bearded whales are probably co-derived traits, but the "tooth" trait of tooth whales is considered a primitive trait. However, only tooth whales have the ability to eco-locate, and the accompanying melon body is one of the most famous traits suggesting monophyly of whales.
[0065]
While the morphological classification strongly insists on the monophyly of whales and bearded whales, most of the phylogenetic analyzes using molecules that have been performed so far indicate that This suggests that no phylogenetic group is formed. Among them, the spark of great controversy was the phylogenetic tree proposed by the comparative analysis of mitochondrial gene sequences of Milinkovith and Meyer (1993). According to this analysis, the sperm whale superfamily group included in tooth whales It is more closely related to bearded whales than other toothed whales (that is, toothed whales are multi-lineage). A number of molecular statistical studies have been conducted following this result, but many have resulted in multi-lineage or unresolvable dental whales (eg, Arnason et al., 1994; Adachi et al. Smith et al., 1996: see FIG. 17). Therefore, species discrimination was performed using the SINE method in order to solve the following problems: Problem (1) Problems of monophyly of cetaceans; (2) Phylogenetic relationships in the order Coleoptera including all dolphins (3) Divergence age of each cetacean. According to the present invention, it was possible to clarify not only the determination of the systematic relationship but also the problems of the statistical analysis which has been carried out with absolute reliability up to now.
[0066]
As described above, the inventor of the present application roughly divided into CHR-2 present in the artiodactyl genome, FL (Full Length), MD (Middle Deletion Type), DT (Deletion Type), and CD (Cetacea Deletion type). It has been discovered that there is a subfamily of CDOs (Cetacea Deletion type Odontoceti). The distribution of each subfamily is thought to reflect a phylogenetic relationship because the amplification stages differ during the evolution process. An alignment of the consensus sequences for each subfamily is shown in FIG. As is apparent from FIG. 18, each subfamily has a diagnostic base substitution or deletion.
[0067]
Dot hybridization was performed to confirm the distribution of these SINE subfamilies in whales and artiodactyla. For the probe used, an oligo probe was designed at a position at which the CD and CDO could be distinguished from each other (thick line of the alignment), and the PCR product was used for FL. FIG. 12 shows the results of dot hybridization. First, it was suggested that FL was distributed only in hippos and cattle (representing ruminants) in all cetaceans and artiodactyla, and was not present in the genomes of mammals added as pigs, camels and other external groups. Next, it is thought that CD is specifically distributed only in the order Coleoptera and not in the even-toothed order, strongly supporting the monophyly of the order Coleoptera. Furthermore, regarding CDO, since a strong signal is confirmed only in tooth whales, this subfamily is thought to have explosively amplified specifically in subspecies of tooth whales. Data supporting single system. However, if no signal is confirmed in the analysis by dot hybridization, it is not possible to deny the possibility that the copy number was simply low in the strain, and therefore the strain relationship cannot be estimated from the result alone. Therefore, the loci actually inserted by the SINE into the genome of the order Coleoptera were isolated and the lineage relationship was estimated.
[0068]
Example 2-1: SINE Of the internal lineage of the order Coleoptera using insertion into the genome
In the study on the internal lineage of Coleoptera, CDs that are specifically distributed throughout the Coleoptera among SINEs present in Coleoptera and CDOs that are thought to specifically amplify in the subspecies of Coleoptera are noted. Then, loci containing SINE sequences belonging to these subfamilies were isolated from various genomic libraries of the order Coleoptera. As described above, for efficient phylogenetic analysis, a prediction can be made by associating the distribution of subfamilies with their amplification times. Regarding the phylogenetic analysis of sperm whales, the reason that such loci could not be isolated despite the continued search for loci suggesting monophyly of whales is mainly It was thought that this was because they were paying attention to CDOs distributed only in. Therefore, only screening of the sperm whale genomic library was carried out using the sequence of the CD which was presumed to have been amplified immediately before the CDO. The correspondence between the names of the loci described below and the whale species used in the genomic library is summarized below: Mago: Magondow, Isi: Dolphin, Amz: Amazon dolphin, Tuti: Beaked whale, Sp and Sperm : Sperm whale, Bando: band dolphin, and Hump: humpback whale. The PCR primers used to isolate loci suggesting a phylogenetic relationship in Coleoptera are shown in Table 3 below:
[Table 3]

Shown in
[0069]
Example 2-2: Locus supporting monophyly of cetaceans
The locus Bando1 was isolated from bottlenose dolphins, and the locus Sp316 from a sperm whale genomic library. The nucleotide sequences of these loci were determined, primers were designed in the vicinity of SINE, PCR was performed using genomic DNA of the artiodactyla as a template, and separation was performed by agarose gel electrophoresis. As a result, as shown in FIG. 19, a band pattern was confirmed at the loci Bando1 and Sp316, which had a SINE insertion commonly in whale eyes. The birch, which was used as the most closely related outer group, did not have this insertion, so it is probable that the insertion occurred when the whale was a common ancestor. These loci strongly support the monophyly of cetaceans. At the locus Bando1, it is probable that CD was first inserted into the common ancestor of the order Coleoptera, followed by insertion of CDO in the common ancestors of two species of dolphins in South America, Amazon dolphin and Laplata dolphin. Thus, this locus suggests not only the monophyly of the cetacean but also the monophyly of South African dolphins. At locus Sp316, it indicates that CHR-1 type III is a common ancestor of the order Coleoptera. 20 to 22 show the results of alignment of locus Bando1, and FIG. 23 shows the results of alignment of locus Sp316.
[0070]
Example 2-3: Locus supporting monophyly of dolphins
At the locus Mago19, insertion of CDO specific to four species of dolphins (Dermatidae: Coleoptera: Aegypti, bottlenose dolphins; Dolphinid: Dolphins; Narwidae: Narwhal) was confirmed (see FIG. 24). This locus strongly supports the monophyly of dolphins. Figures 25 and 26 show the results of the alignment of the locus Mago19.
[0071]
Example 2-4: Infraorder: Delphinida Loci that support
At loci Mago24, Mago26, Mago32, Isi14, Isi36, and Isi38, insertion of CDO was confirmed only in four species of dolphin superfamily, two species of South American dolphins, Amazon dolphin, Laplata dolphin, and Yousko dolphin (see FIGS. 27 and 28). Thus, these loci support the monophyly of this group. According to Muizon (1988, 1994), these groups are grouped as Delphinida lower order (order lower than suborder) based on morphological characteristics, but this idea is well consistent. This is strongly supported in the analysis by the sequence of the mitochondrial Cytb gene of Arnason (1996). It also suggests that the dolphins are a multi-lineage group and that Ganges dolphins are more primitive than other dolphins. FIG. 29 shows the result of alignment of the locus Ishi14. 30 to 31 show the results of alignment of the locus Ishi36. 32 to 33 show the results of alignment of the locus Ishi38. 34 to 35 show the results of alignment of the locus Mago24. 36 to 37 show the results of alignment of the locus Mago26. 38 to 39 show the results of alignment of the locus Mago32.
[0072]
Example 2-5: Locus indicating phylogenetic relationship of red whales
At the locus Mago8 and Mago13, insertion of CDOs specific to Delphinida and two species of ape and whale belonging to the family Spermaceae was confirmed (see FIG. 40). From this, it can be said that among the current species, the red whale family is the most closely related taxon to Delphinida. It can also be seen that Ganges dolphins are a more primitive group than red whales. FIG. 41 shows the result of alignment of the locus Mago8. FIG. 42 shows the result of alignment of the locus Mago13.
[0073]
Example 2-6: Gene locus suggesting a phylogenetic relationship of Ganges dolphinaceae
At the locus Mago21 and Mago22, insertion of CDOs specific to Delphinida, red whale family and Ganges dolphin was confirmed (see FIG. 43). Thus, the Ganges dolphins (Indus dolphins are not included in the analysis due to lack of samples, but probably form a monophyletic group with Ganges dolphins) may be the most primitive group of tooth whales except sperm whales It became clear. FIG. 44 shows the result of alignment of the locus Mago21. FIGS. 45-47 show the alignment results for the locus Mago22.
[0074]
Example 2-7: Gene loci supporting monophyly of whales
At loci Sperm8, Sperm28, and Sperm47, insertion of CD was confirmed in all species of the order Ceratidae including sperm whales (see FIG. 48). Analysis of these loci strongly supported the monophyly of whales, which have been questioned by molecular statistical studies. 49 to 50 show the results of alignment of the gene locus Sperm8. FIG. 51 shows the alignment result of the gene locus Sperm28. FIG. 52 shows the result of alignment of the gene locus Sperm47.
[0075]
Example 2-8: Gene loci supporting monophyly of dolphins in South America
At loci Amz13 and Bando1, CDO insertion was commonly observed in two species of South American dolphins, Amazon dolphin and Laplata dolphin (see FIG. 53). There are several hypotheses regarding the phylogenetic relationship between the two species of South American dolphins and the three species of dolphins, and that the dolphins form a monophyletic group with any of the dolphins in South America (Kasuya, 1973; Barnes et al.). al., 1985) These two loci strongly supported that the South American dolphins are monophylic. Among the several hypotheses that have been proposed so far, it is considered that this is the most ideal phylogenetic relationship in biogeography. FIG. 54 shows the result of alignment of the locus Amz13.
[0076]
Example 2-9: Locus specific for Amazon dolphins Amz11
FIG. 55 shows the band pattern of the locus Amz11 indicating that CDO was specifically inserted into the Amazon dolphin. FIG. 56 shows the alignment result of the locus Amz11.
[0077]
Example 2-10: Locus specific for red whale family Tuti24, Tuti35
FIG. 57 shows the band patterns of loci Tuti24 and Tuti35 indicating that CDO was specifically inserted into the red whale family. FIG. 58 shows the result of alignment of the locus Tuti24. FIGS. 59-60 show the results of alignment of Tuti35 locus.
[0078]
Example 2-11: Gene-specific loci for three sperm whales Sp9 And sperm whale-specific Sp2
FIG. 61 shows the locus Sp9 indicating that CDO was inserted into three species of Sperm whales, and the band pattern of Sp2 specific to Sperm whales. 62 to 63 show the results of alignment of the gene locus Sp2. FIG. 64 shows the result of alignment of the locus Sp9.
[0079]
Example 2-12: Locus specific to fin whales (or bearded whales) Hump20, Hump203
FIG. 65 shows the band patterns of the lump Hump20 and Hump203, which indicate that a CD specific to the fin whale family (or bearded whale) was inserted. 66 to 68 show the results of alignment of the locus Hump20. FIG. 69 shows the result of alignment of the locus Hump203.
[0080]
Example 3: Creation of a phylogenetic tree inside Whale
The SINE insertion patterns obtained from the above results are summarized in a matrix (see FIG. 70). FIG. 71 shows a phylogenetic tree inside the whales estimated based on this.
The nucleotide sequences of the PCR products obtained at the time of analysis of each locus were all determined and aligned as much as possible. It was confirmed that the resulting band pattern was not caused by secondary insertion or deletion. We also believe that these sequences will be important for future research as nuclear gene sequence information for whales.
[0081]
result
The phylogenetic relationships of modern cetaceans as revealed by the above examples are summarized below. 1. Dental whales form a monophyletic group. The sperm whale superfamily is the first branch of the modern dentate whale.
2. Ganges dolphins (probably Indus dolphins and a single line) diverged next to sperm whales. It is derived from a different family from other dolphins.
3. Next, the red whale family diverges. The genus Beradius and the genus Mesoprodon of the family Red Whale are a single line.
4. Two species of dolphins in South America, Amazon dolphin and Laplata dolphin, are single strains.
5. Dolphins, porpoises and narwhals belonging to the dolphin family form a monophyletic group.
[0082]
The above phylogenetic relationship is not only phylogenetically significant, but also when a sample consisting of several animals is provided, by selecting an appropriate locus in which SINE has been inserted, the base sequence can be determined. This forms the basis of the reliability of the species discrimination method using the SINE method according to the present invention, which can discriminate which specimen the specimen is without analysis. By establishing a phylogenetic tree for animals other than whales by the SINE method, a species discrimination method similar to that in the whales can be established in those eyes. This is because phylogenetic analysis using molecular statistical techniques has limitations as described below.
In the past, molecular statistical studies on phylogenetic relationships in sperm whales have yielded few results suggestive of monophyly in tooth whales. Since these analyzes mainly used the sequence of the mitochondrial gene, it was initially thought that this was a phenomenon specific to mitochondria. However, the same results were obtained in the analysis of the amino acid sequence of myoglobin (Czelusniak et al., 1990). In addition, analysis of the nuclear gene IRBP (Smith et al., 1996) shows that the multilineage of tooth whales (sperm whales are more closely related to baleen whales) is statistically advantageous. From this, it seems that statistical analysis of the molecular sequence would give a result such that the tooth whale is multi-lineage (see FIG. 72). However, the above results clearly suggest that whales form a monophyletic group, and morphological characteristics suggest that whales are monophylic.
[0083]
So why would molecular statistical analysis of whales result in sperm whales being more closely related to baleen whales? Often the issue is the choice of outgroups. In other words, only molecular distance of each group is known from the molecular information, and the roots of the phylogenetic tree (Root) are obtained by using the outer group branched before the group (in this case, dolphin, sperm whale, and baleen whale) branch. ) Needs to be determined. However, the position may differ depending on the type and number of the outer group, and an analysis in which the outer group uses only one type of cattle, such as the analysis of the earlier Arnason (1994), is considered to be considerably less reliable. In fact, according to the study of Adachi and Hasegawa (1995) regarding the Cytb gene, it has been found that changing the outgroup greatly changes the results. However, subsequent analyzes using a large number of artiodactyla and other ungulates as the outer group (Arnason et al., 1996) failed to suggest monophylogeny of dendrites, and thus were simply outgrouped. It is unlikely that the choice was a problem. Next, it is possible that the rate of molecular evolution differs between taxonomic groups. The study of Martin (1993) et al. Shows a correlation between the influence of metabolism, which is thought to affect the rate of base substitution, and the size of the body of an organism. In general, the larger the body size, the slower the rate of base substitution. Suggested that. In other words, it is considered that small-sized tooth whales such as dolphins caused a statistical error because their molecular evolution rate was higher than that of large bearded whales such as sperm whales and fin whales. However, the maximum likelihood analysis (Maximum likelihood analysis) finally used by Hasegawa (1997) for analysis is different from the maximum parsimony analysis (Maximum parsimony analysis). As a result, the result was not changed despite the fact that it is rare that a phylogenetic tree was made incorrectly.
[0084]
It is also quite possible that seed branching occurred in a short time. For example, if the sperm whales diverge from the cetacean in a very short time after the divergence of the cetaceans, it is not sufficient for the common base substitution to accumulate between the cetaceans. If there is no time, and if sperm whales have their own history for a long time until the present, the base substitution accumulated during that time will be a little common that tooth whales would have accumulated during the period of common ancestry It is considered that the base substitution is neglected. Indeed, speciation through adaptive radiation of whales is expected to have taken place at a considerable rate due to the establishment of the Antarctic Current caused by the northern deviation of the Australian continent about 35 million years ago (Fordyce, 1992). ). In such a case, it is considered that systematic estimation by molecular statistical analysis is impossible using any analysis method. Also, even if a more probable phylogenetic estimation could be made on the basis of various statistical assumptions, it would result in a rather artificial phylogenetic tree. This seems to deviate from the notion of "objectivity", which is superior to morphological research.
[0085]
【The invention's effect】
One of the advantageous effects of the method for determining an animal species according to the present invention is that the sequencing step can be omitted. In general, a sequencer costs at least 15 million yen, and for consumables, the cost of a single sequence is approximately 1200 yen. Therefore, when analyzing a large number of samples, it is very expensive. Furthermore, in the case of a commercial analysis by a test company, at present, a charge of 10,000 yen or more per sample is charged. In terms of time, the sequence determination step requires at least about one and a half hours per sample, and the species discrimination method using the SINE method, which can omit this, has high analysis efficiency. Most importantly, DNA sequence analysis involves statistical manipulations. As described above, such a statistical method has a problem in that the result differs depending on the analysis method. On the other hand, in the SINE method, the species is determined using the irreversible phenomenon of SINE insertion as an index, so that the result is clear and no special statistical processing is required. In conclusion, the species discrimination method using the SINE method according to the present invention is superior to the conventional species discrimination method based on comparison of nucleotide sequences in terms of money, time, and reliability. I can say.
[0086]
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[0087]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a general diagram for the SINE retroposition.
FIG. 2 (A) represents a master gene model, and FIG. 2 (B) represents a multi-source gene model.
FIG. 3 shows the utility of SINE as an indicator of evolution.
FIG. 4 shows a phylogenetic tree model of species AD. FIG. 4C shows a PCR pattern.
FIG. 5 is an example of some patterns of transcripts from total genomic DNA from selected animal species.
FIG. 6 shows the tRNA-like structure of CHR-2 SINE.
FIG. 7 (A) shows the tRNA-like secondary structure of CHR-2 SINE, and FIG. 7 (B) shows the secondary structure of human tRNA Glu.
FIG. 8 depicts the construction of a CHR-2 SINE consensus sequence from several sequence alignments of CHR-2.
FIG. 9 is a phylogenetic tree including an old SINE amplified at t and a young SINE amplified at u.
FIG. 10 depicts the identification of characteristic nucleotides or possible deletions representing subfamilies of the SINE family by alignment.
FIG. 11 shows an alignment of consensus sequences for subfamilies of the CHR-2 SINE family.
FIG. 12 shows the results of dot-hybridization experiments using probes specific for CD, CDO, and other subfamilies, respectively.
FIG. 13 schematically shows the principle of flanking SINE PCR.
FIG. 14 shows an example of a flanking PCR result. 14 (B) and (C) simultaneously show the results of a hybridization experiment performed using the same filter using two different probes.
FIG. 15 shows a mammalian phylogenetic tree.
FIG. 16 shows a flow of an experiment in the SINE method.
FIG. 17 shows a phylogenetic tree (controversy over the problem of monophyly of cetaceans) proposed from molecular statistical studies.
FIG. 18 shows an alignment of consensus sequences for each subfamily of CHR-2.
FIG. 19 shows the band patterns of loci Bando1 and Sp316 indicating that SINE was commonly inserted in the order of Whales.
FIG. 20 shows the results of alignment of the locus Bando1.
FIG. 21 shows the results of alignment of the locus Bando1.
FIG. 22 shows the results of alignment of the locus Bando1.
FIG. 23 shows the results of alignment of the locus Sp316.
FIG. 24 shows the band pattern of the locus Mago19, which indicates that there was a specific CDO insertion in four dolphin superfamily species (Dolphinidae: Scarabaeidae, bottlenose dolphins; porpoises; This locus strongly supports the monophyly of dolphins.
FIG. 25 shows the results of alignment of the locus Mago19.
FIG. 26 shows the results of alignment of the locus Mago19.
FIG. 27 shows the band patterns of the loci Isi14, Isi36, and Isi38, which indicate that CDO was inserted into four species of dolphins, four species of dolphins, two species of dolphins in South America;
FIG. 28 shows the band patterns of loci Mago24, Mago26, and Mago32 indicating that CDO was inserted into four species of dolphins, four species of dolphins, two species of dolphins in South America;
FIG. 29 shows the results of alignment of the locus Ishi14.
FIG. 30 shows the results of alignment of the locus Ishi36.
FIG. 31 shows the results of alignment of the locus Ishi36.
FIG. 32 shows the results of alignment of the locus Ishi38.
FIG. 33 shows the results of alignment of the locus Ishi38.
FIG. 34 shows the results of alignment of the locus Mago24.
FIG. 35 shows the results of alignment of the locus Mago24.
FIG. 36 shows the results of alignment of the locus Mago26.
FIG. 37 shows the results of alignment of the locus Mago26.
FIG. 38 shows the results of alignment of the locus Mago32.
FIG. 39 shows the results of alignment of the locus Mago32.
FIG. 40 shows the band patterns of loci Mago8 and Mago13 indicating that specific CDOs were inserted into Delphinida and two species of ape and whale that belong to the family Spermaceae.
FIG. 41 shows the results of alignment of the locus Mago8.
FIG. 42 shows the results of alignment of the locus Mago13.
FIG. 43 shows the band pattern of the locus Mago21, Mago22 indicating that there was a CDO insertion specific for Delphinida, red whale family and Ganges dolphin.
FIG. 44 shows the results of alignment of the locus Mago21.
FIG. 45 shows the results of alignment of the locus Mago22.
FIG. 46 shows the results of alignment of the locus Mago22.
FIG. 47 shows the results of alignment of the locus Mago22.
FIG. 48 shows the band patterns of the loci Sperm8, Sperm28, and Sperm47, which indicate that CD was commonly inserted in all species of the subclinical order, including sperm whales.
FIG. 49 shows the results of alignment of the gene locus Sperm8.
FIG. 50 shows the results of alignment of the Sperm8 locus.
FIG. 51 shows the results of alignment of the Sperm28 locus.
FIG. 52 shows the results of alignment of the Sperm47 locus.
FIG. 53 shows the band patterns of the loci Amz13 and Bando1 indicating that CDO was commonly inserted in two species of South American dolphins, Amazon dolphin and Laplata dolphin.
FIG. 54 shows the results of alignment of the locus Amz13.
FIG. 55 shows the band pattern of the locus Amz11 indicating that CDO was specifically inserted into the Amazon dolphin.
FIG. 56 shows the results of alignment of the locus Amz11.
FIG. 57 shows a band pattern of loci Tuti24 and Tti35 indicating that CDO was specifically inserted into the red whale family.
FIG. 58 shows the results of alignment of the Tuti24 locus.
FIG. 59 shows the results of alignment of the Tuti35 locus.
FIG. 60 shows the results of alignment of the locus Tuti35.
FIG. 61 shows the band pattern of Sp9, a locus indicating that CDO was inserted into three species of Sperm whales, and Sp2 specific to Sperm whales.
FIG. 62 shows the results of alignment of the locus Sp2.
FIG. 63 shows the results of alignment of the locus Sp2.
FIG. 64 shows the results of alignment of the locus Sp9.
FIG. 65 shows the band patterns of the loci Hump20 and Hump203, which indicate the insertion of a CD specific to the fin whale family (or bearded whale).
FIG. 66 shows the results of alignment of the Hump20 locus.
FIG. 67 shows the results of alignment of the Hump20 locus.
FIG. 68 shows the results of alignment of the Hump20 locus.
FIG. 69 shows the results of alignment of the Hump203 locus.
FIG. 70 shows a matrix in which SINE insertion patterns are summarized.
FIG. 71 shows a phylogenetic tree in the order of Coleoptera estimated based on the matrix shown in FIG. 70.
FIG. 72 shows the results (limits) of molecular statistical analysis.

Claims (25)

A method for discriminating among species including a bottlenose dolphin, a humpback whale, a fin whale, a fin whale, a fin whale and a fin whale, a sperm whale, a humpback whale, and a fin whale, a sperm whale, a humpback whale, a ganji swelling dolphin, a sperm whale, a giant whale, a fin whale, a fin whale, a fin whale, a fin whale, a fin whale, and a fin whale There are the following steps:
Creating a genomic DNA library from one of the above whales to be identified;
Isolating from said library a clone containing an orthologous locus comprising a sequence selected from the group consisting of the CD sequence shown in SEQ ID NO: 6 and the CDO sequence shown in SEQ ID NO: 7;
Amplifying by PCR the sequence at the locus for the species using a set of PCR primers that anneal to flanking sequences located on either side of the CD or CDO sequence at the locus; Gel electrophoresis to determine the species by the presence or absence of a band indicating the presence of the locus;
The method, comprising: The orthologous locus is Bando1, which corresponds to the sequence shown in any one of SEQ ID NOs: 8 to 21, and its presence in the gel electrophoresis indicates that one species of the cetacean to be discriminated is Amazon dolphin Or the method of claim 1, wherein the method is indicative of Laplata dolphin. The orthologous locus is Mago19 corresponding to the sequence shown in any one of SEQ ID NOs: 36 to 46, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a Pacific dolphin 2. The method of claim 1, wherein the method is indicative of being a dolphin, a dolphin or a narwhal. The orthologous locus is Ishi14 corresponding to the sequence shown in any one of SEQ ID NOs: 47 to 51, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a bottlenose dolphin 2. The method of claim 1, wherein the method is indicative of being a dolphin, a dolphin, a porpoise, a narwhal, a northern dolphin, an Amazon dolphin, or a laplata dolphin. The orthologous locus is Ishi36 corresponding to the sequence shown in any one of SEQ ID NOS: 52 to 65, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a Pacific dolphin 2. The method of claim 1, wherein the method is indicative of being a dolphin, a dolphin, a porpoise, a narwhal, a northern dolphin, an Amazon dolphin, or a laplata dolphin. The orthologous locus is Ishi38 corresponding to the sequence shown in any one of SEQ ID NOs: 66 to 79, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a Pacific dolphin 2. The method of claim 1, wherein the method is indicative of being a dolphin, a dolphin, a porpoise, a narwhal, a northern dolphin, an Amazon dolphin, or a laplata dolphin. The orthologous locus is Mago24 corresponding to the sequence shown in any one of SEQ ID NOs: 211 to 224, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a bottlenose dolphin 2. The method of claim 1, wherein the method is indicative of being a dolphin, a dolphin, a porpoise, a narwhal, a northern dolphin, an Amazon dolphin, or a laplata dolphin. The orthologous locus is Mago26 corresponding to the sequence shown in any one of SEQ ID NOs: 225 to 238, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a bottlenose dolphin 2. The method of claim 1, wherein the method is indicative of being a dolphin, a dolphin, a porpoise, a narwhal, a northern dolphin, an Amazon dolphin, or a laplata dolphin. The orthologous locus is Mago32 corresponding to the sequence shown in any one of SEQ ID NOs: 80 to 93, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a bottlenose dolphin 2. The method of claim 1, wherein the method is indicative of being a dolphin, a dolphin, a porpoise, a narwhal, a northern dolphin, an Amazon dolphin, or a laplata dolphin. The orthologous locus is Mago8 corresponding to the sequence shown in any one of SEQ ID NOs: 94 to 98, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a Pacific dolphin The method of claim 1, wherein the method is indicative of being a dolphin, a dolphin, a porpoise, a narwhal, a dolphin, a dolphin, an Amazon dolphin, a laplata dolphin, a beaked whale, or a giant whale. The orthologous locus is Mago13 corresponding to the sequence shown in any one of SEQ ID NOs: 99 to 112, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a bottlenose dolphin The method of claim 1, wherein the method is indicative of being a dolphin, a dolphin, a porpoise, a narwhal, a dolphin, a dolphin, an Amazon dolphin, a laplata dolphin, a beaked whale, or a giant whale. The orthologous locus is Mago21 corresponding to the sequence shown in any one of SEQ ID NOS: 113 to 117, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a bottlenose dolphin The method of claim 1, wherein the method is indicative of being a dolphin, a dolphin, a porpoise, a narwhal, a dolphin, a dolphin, an Amazon dolphin, a La Plata dolphin, a beaked whale, a giant whale, or a Ganges dolphin. The orthologous locus is Mago22 corresponding to the sequence shown in any one of SEQ ID NOs: 239 to 252, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a bottlenose dolphin The method of claim 1, wherein the method is indicative of being a dolphin, a dolphin, a porpoise, a narwhal, a dolphin, a dolphin, an Amazon dolphin, a La Plata dolphin, a beaked whale, a giant whale, or a Ganges dolphin. The orthologous locus is Sperm8 corresponding to the sequence shown in any one of SEQ ID NOs: 118 to 129, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a bottlenose dolphin The method of claim 1, wherein the indicator indicates that the species is a dolphin, a dolphin, a porpoise, a narwhal, a dolphin, a dolphin, an Amazon dolphin, a Laplata dolphin, a beaked whale, a giant whale, a Ganges dolphin or a sperm whale. The orthologous locus is Sperm28 corresponding to the sequence shown in any one of SEQ ID NOS: 130 to 135, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a Pacific dolphin The method of claim 1, wherein the indicator indicates that the animal is a dolphin, a dolphin, a porpoise, a narwhal, a dolphin, a dolphin, an Amazon dolphin, a laplata dolphin, a beaked whale, a giant whale, a Ganges dolphin or a sperm whale. The orthologous locus is Sperm47 corresponding to the sequence shown in any one of SEQ ID NOs: 136 to 140, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a bottlenose dolphin The method of claim 1, wherein the indicator indicates that the species is a dolphin, a dolphin, a porpoise, a narwhal, a dolphin, a dolphin, an Amazon dolphin, a Laplata dolphin, a beaked whale, a giant whale, a Ganges dolphin or a sperm whale. The orthologous locus is Amz13 corresponding to the sequence shown in any one of SEQ ID NOs: 141 to 145, and its presence in the gel electrophoresis indicates that one species of the cetacean species to be discriminated is Amazon dolphin Or the method of claim 1, wherein the method is indicative of Laplata dolphin. The orthologous locus is Amz11 corresponding to the sequence shown in any one of SEQ ID NOs: 146 to 151, and its presence in the gel electrophoresis indicates that one species of the cetacean to be discriminated is Amazon dolphin The method of claim 1, wherein the method indicates: The orthologous locus is Tuti24 corresponding to the sequence shown in any one of SEQ ID NOs: 152 to 156, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a sperm whale or The method of claim 1, wherein the method is indicative of a giant whale. The orthologous locus is Tuti35 corresponding to the sequence shown in any one of SEQ ID NOs: 157 to 171, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a sperm whale or The method of claim 1, wherein the method is indicative of a giant whale. The orthologous locus is Sp2 corresponding to the sequence shown in any one of SEQ ID NOs: 172 to 185, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a sperm whale The method of claim 1, wherein the method indicates: The orthologous locus is Sp9 corresponding to the sequence shown in any one of SEQ ID NOs: 186 to 190, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is one of the sperm whales The method of claim 1, wherein the method indicates: The orthologous locus is Hump20 corresponding to the sequence shown in any one of SEQ ID NOs: 191 to 204, and its presence in the gel electrophoresis indicates that one of the whales to be identified is a humpback whale. The method of claim 1, wherein the method is indicative of being a fin or minke whale. The orthologous locus is Hump203 corresponding to the sequence shown in any one of SEQ ID NOS: 205 to 210, and its presence in the gel electrophoresis indicates that one of the whales to be discriminated is a humpback whale. The method of claim 1, wherein the method is indicative of being a fin or minke whale. 2. The method of claim 1, wherein said PCR primer is selected from the group consisting of SEQ ID NOs: 276-325.

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