JP4246262B2 - PIT-1 gene polymorphism and animal trait selection - Google Patents

Description

制限酵素消化による該13-kbインサートのXhoI、HinfI、及びEcoRIサブクローンの特性決定、及び配列決定によって、該クローンがウシPit-1ゲノム断片として同定された。
同様に、ウシ以外の様々なゲノムライブラリー中にPit-1遺伝子を同定するために、上記のWoolardらが教示する方法を用いることができる。これは、以下に記載するように、様々な動物から該配列を増幅するために用いることができるPit-1ゲノム断片内の特異的な配列の同定を可能にするであろう。
動物のPit-1遺伝子中の突然変異を同定する第一のステップは、動物から精液、血液、細胞、生検組織、糞などのサンプル（これらに限定されない）を得ることである。次に、Sambrookらの、「Molecular Cloning, A Laboratory Manual, second edition, 1989」に記載されているような、本分野で公知の方法を用いて得られた試料から、ゲノムDNAを抽出し得る。
しかし、精液についてはWalshの、Biotechniques, 10:506(1991)に記載されている操作、血液についてはLewin及びStewart-HaynesのBiotechniques, 13:522に記載されている操作を用いてゲノムDNAを抽出することが好ましい。
ゲノムDNAを抽出した後、本分野には、Pit-1遺伝子中の突然変異を検出するための公知の方法がいくつか存在する。任意の検出方法を、突然変異を検出するために利用することができる。これらの方法の例には、RFLP、SSCP、DGGE、CFLP、及びProsser、Trends Biotech 11:238-246(1993)、及び上記Sambrookらによって記載されているような一塩基変異が含まれるが、これに限定されない。これらの方法は、以下でさらに詳細に論述する。
例えば、RFLP法では、PCRプライマーは、標準的な操作によって、Pit-1遺伝子を含む断片を増幅するために使用される。Pit-1配列の増幅を可能にする任意のPCRプライマーを利用することができ、このようなプライマーを同定する特定のクローンを単離する方法を利用できる。
本発明の好適な態様では、PCRプライマーは、上記のWoolardらによって記載された451-bp断片のようなPit-1遺伝子の多型を含有する断片のイントロンＶとエキソン６からデザインすることができる。さらに好適な本発明の態様では、PCRプライマーは以下のとおりである。

Pit-1断片の増幅は、上記Sambrookらに記載されているような、標準的PCR操作を用いて行い得る。しかし、2mM MgCl₂を含有する50μＬ反応容量の中でゲノムDNAを増幅することが好ましい。
本発明の好適な態様では、88〜98℃で10〜15分間、90〜100℃で約１分間の後、90〜100℃で20〜40秒間25〜50サイクル；40〜60℃で１〜５分間；68〜80℃で約１〜５分間というPCR反応条件を用いることができる。最後のステップには、68〜80℃で８〜12分間というサイクルが含まれ得る。
増幅後、続いて、様々な制限酵素又は本分野で公知のエンドヌクレアーゼを用いてPit-1中の特定の突然変異を切断する。これらの制限酵素には、BamHI、EcoRI、SmaI、HinfI等が含まれるが、これらに限定されない。例えば、上記のSamrookらに記載されている酵素を参照されたい。Pit-1遺伝子の突然変異した対立遺伝子を切断し、Pit-1遺伝子の突然変異していない対立遺伝子を切断しない制限エンドヌクレアーゼを用いることは、特に興味深い。本発明の好適な態様では、動物のミルク産生、脂肪、タンパク質、及び筋肉性形質の同定に関しては、HinfIが用いられる。
消化後、続いて、該サンプルをアガロースゲル上で電気泳動し、例えば臭化エチジウムのような染色剤（しかし、断片を同定する任意の染色剤を用いることができる）を用いて同定する。
SCCP（一本鎖DNA高次構造多型；single stranded conformation poly-morphism）も、単離されたゲノムPit-1断片中の一又は複数の突然変異を同定し得る、本分野で公知の方法である。該方法は、上述したプライマーと類似のプライマーを用いるPCR増幅に基づいている。次に、増幅された断片を³²Pのような標識又は他の任意の適切な放射性標識でラベルする。次に、例えば加熱後急冷することによって、該放射性標識された断片を変性させる。冷却後、非変性技術を用いて該断片を電気泳動し、次にオートラジオグラフ（audioradiographed）にかける。
DGGE（denaturing gradient gel electrophoresis）は、Pit-1突然変異を検出するための、さらに別の方法である。該方法では、断片は、上述したような適切なプライマーを用いるPCRによって増幅され、変性グラジエントにかけられる。さらに、該サンプルを電気泳動して、突然変異を検出する。
突然変異を検出するのに使用し得るさらに別の方法は、CFLPである(切断断片長多型；cleavage fragment length polymorphism）。該方法は、野生型DNAと変異型DNAの二つの分子の間で、DNA配列中に唯一つの塩基が突然変異しているのを検出することができる。該方法は、現在ベーリンガー・マンハイムによって市販され、キットの形態で購入することができる。
Pit-1遺伝子中の突然変異を検出するために使用し得る別の方法は、Pit-1遺伝子の突然変異した領域をカバーするプライマーを利用する。
より好ましくは、二組のプライマー（突然変異したPit-1遺伝子をカバーし、これに特異的なプライマーを含有する組、突然変異していないPit-1遺伝子をカバーし、これに特異的なプライマーを含有する別の組）を用いて、抽出したゲノムDNAサンプルに対して、二つの別個の増幅反応を行う。より好ましくは、用いるプライマーは、増幅産物を容易に可視化し得るように標識されている。該方法によれば、テストしたゲノムDNAが、ホモ接合の突然変異Pit-1遺伝子（二つの突然変異した対立遺伝子）を含有するときには、突然変異領域に特異的なプローブを用いる増幅反応だけがシグナル（すなわち、増幅産物）を生じるであろう。テストしたゲノムDNAがヘテロ接合（すなわち、一つが突然変異対立遺伝子、一つが非突然変異対立遺伝子）である時には、二つの増殖反応がシグナル（増幅産物）を生じるであろう。同様に、テストしたゲノムDNAがホモ接合の非突然変異Pit-1遺伝子を含有する時には、非突然変異領域に特異的なプローブを用いる増幅反応だけがシグナルを生じるであろう。それ故、単一の増幅ステップで、テストしたDNAの対立遺伝子のパターンが明らかとなる。
別の態様では、WO 97/06276に記載されている技法の使用は、マーカーのホモ接合状態又はヘテロ接合状態を単一ステップで検出するのに特に適する。
これらの方法では、様々なパターンの中から識別するために、増幅された産物をさらに切断する必要がない。さらに、検出ステップの前に、特異的なPit-1遺伝子断片を増幅する必要もない。最後に、用いたラベルの性質によっては、可視化はきわめて容易であり得る。例えば、プローブが放射線ラベルされている場合には、プローブは電気泳動によって得られる。より興味深いことには、プローブを染色剤でラベルすると、直接可視化が得られる。
第一の態様では、テストは、プレートのような極めて単純な装置中で実施し得る。ゲノムDNAのサンプルを二つのウェル中（一方にはPit-1遺伝子の突然変異をカバーし、突然変異したPit-1遺伝子に特異的な一つのプローブを含有するラベルされたプローブの組が入っており、他方にはPit-1遺伝子突然変異をカバーし、非突然変異Pit-1遺伝子に特異的な一つのプローブを含有するプローブの組が入っている）に導入する。
増幅後、標識が直接プレート中に現れ、自動化された装置によって分析することができる。
該増幅法では、プローブの各組で用いられる第二のプライマーは、200〜400bp、さらに好ましくは320〜370bpを含有する産物の増幅を可能とするように選択する。このような第二のプライマーは、例えば以下のプライマーから選択することができる。

各プライマーの長さは、好ましくは20〜40塩基、より好ましくは25〜35を具備する。
該戦略のための適切なプローブの選択は、発明者が、観察された多型の原因となるPit-1遺伝子中の突然変異を同定することによって可能となる。さらに特異的には、該突然変異は、Pit-1コード領域中、1178位のヌクレオチドに生じ、突然変異した該遺伝子中ではアデニンがグアニンに置き換わっている。該突然変異は、図２に示されている。
突然変異した領域（突然変異）をカバーするプローブの位置は、変わり得る。
さらに好ましくは、第一の態様では、プライマーの二組のプライマーは、ミルク産生能の特徴となるAA遺伝子型については、

肉産生能の特徴となるBB遺伝子型については、

である。
別の態様では、WO 97/06276を用いる時には、
種々のサイズの増幅された断片を産生せしめる二組のプライマーは、
ミルク産生脳の特徴となるAA遺伝子型については、

であり、第二のプライマーPit-1 C’は、肉産生脳の特徴であるBB遺伝子型が、Pit-1 AA及びPit-1Bを用いて得られたものに比べて少なくとも10bp以上短く又は長く増幅されるように選択する。
多くの突然変異検出法が利用できるが、本発明は上記で論じた方法に限定されず、突然変異を検出する全ての方法が含まれる。
続いて、対立遺伝子及び対立遺伝子パターンを同定し、次に対立遺伝子を同定することによって明らかとなる特異的な形質を決定するために統計的な分析を行う。さらに具体的には、娘収量偏差（DYD；daughter yield deviation）及び脱回帰プルーフ（DRP；deregressed proof）を同定し得る任意の統計プログラムを利用し得る。SASのMIXED操作を用いて統計分析を行うことが好ましい（ユーザー用ガイド、Statistics, Version 6,4th ed. SAS Inst., Inc. Cary, N.C.(1990)）。Technical Report P 229 SAS Inst., Inc., Cary, N.C.(1992)。本発明で用いた統計分析は、以下の例で詳述する。
本発明には、ゲノムDNAの抽出用材料、配列番号１及び２（上述）を有するPCRプライマー、電気泳動ゲル等の突然変異を可視化するのに必要な材料を含有するキットも含まれる。キットの内容は、用いる検出法に応じて変わってもよく、これについては詳しく先述する。
Pit-1遺伝子中の突然変異をカバーするプライマーも本発明に含まれる。
より具体的には、本発明は、一つの突然変異を有するPit-1遺伝子の領域と相補的な20〜40塩基を具備するプライマーにも関する。
本発明には、Pit-1遺伝子中の200〜400塩基の領域の増幅を可能とするプライマーの組も含まれ、前記領域は一つの突然変異を含有している。
以下のプライマーが本発明に含まれる。

本発明及びその利点をさらに説明するために、以下の具体例を挙げるが、単なる例示であって、決して限定を加えるものではないことを理解されたい。
例Ａ
１．DNAの抽出とPCR
Lucyら、Domest. Anim. Endocrinol. 10:325(1993)によって記載されているように、市販の登録された89匹のイタリアンホルスタイン−フリージアン牛のゲノムDNAを精液から抽出した。
HinfI制限酵素を用いたPit-1遺伝子のRFLPが、上記のWoolardらに適合させたPCR分析によって明らかとなった。
PCRプライマーをイントロンＶとエキソン６からデザインした。用いたプライマーの配列は、5’-AAACCATCATCTCCCTTCTT-3’（配列番号１）及び5’-AATGTACAATGTGCCTTCTGAG-3’（配列番号２）であった。これらのプライマーは、標準的操作によって、2mM MgCl₂を含有する50μＬ反応量中のゲノムDNAから451bp断片を増幅するのに用いた。条件は、94.5℃で10分、94℃で１分の後、95℃で30秒、56℃で１分、72℃で２分を35サイクルであった。最後のステップは、72℃10分であった。PCR産物をHinfIで消化し、１μg/mLの臭化エチジウムを用いて、２%のアガロースゲル上で電気泳動した（図１）。
1996年3月に算出した娘収量偏差(DYD)は、イタリアンホルスタイン−フリージアン・ブリーダー協会ANAFI（Associazione Nazionale Allevatori Frison Italiana, Cremona, Italy）から入手したホルスタイン−フリージアン牛から得た。DYD値は、脂肪とタンパク質のパーセンテージについては算出しない。これらの形質は、収量形質用の解及びこれらの形質に対する平均集団値から間接的に評価し得るにすぎないからである。それ故、DYD値は、パーセンテージ形質に対する遺伝子価（genetic value）の計算法と同一のアプローチを用いて算出した。
同様のDYDは、タイプ形質（type trait）についても利用できなかった。それ故、遺伝子価は、脱回帰プルーフに転換した（Banosら、Interbull Annual Meeting, Aarhus, Denmark, Bulletin No.8, 1993, sigbjornら、J. Dairy Sci, 78:2047(1995)）が、これはDYDを近似していると考え得る。
ウシのサンプルのミルク産生形質に対するDYD及び／又はコンフォメーション形質のDRPの平均及び標準偏差を表１に示す。収量形質（yield trait）に対しては、有効数の娘（分布について調整した群中の娘の数の測定値）が得られたが、タイプ形質に対しては得られなかった。それ故、以下の式：
「有効数」＝「真の数」×「群れの数と娘の数の比の平方根」
を用いて近似した。

2.統計分析
上記SASのMIXED操作を用いて、統計分析を行った。用いた混合モデルは、
y=Xb+Zu+e
ここで、y=ウシのDYD又はDRPのベクトル；b=Pit-1パターンに伴う固定された効果のベクトル；u=ウシのランダム相加的ポリジーン効果のベクトル、及びe=これ以外のランダムな効果のベクトルであった。該モデルは、以下の混合モデル式を用いて解いた。

ここで、Ａは既知の全血縁関係を用いて構築した89匹のウシの間の相加関係行列（既知の祖先1842匹）、R^-1＝D/δ² _e（ここで、Ｄは、対角上に存在する全てのウシに対する有効娘数の対角行列とする）である。次に、残差の分散（residual variance）δ² _eの推定値で該行列を除する。これは、推定されたREMLであり（Patterson and Thompson, Biometrika 58:545 91971）、１ラウンドで収束が起こるので、これは非相互作用性最少分散二次非バイアス化推定(non-interactive minimum variance quadratic unbiased estimation)(Rao, J. Mult. Anal. 1:445(1971))と同一である。得られた推定値は、サンプリングの分散を最少にする二次形態の特性を有する。遺伝率が0.50であると仮定した脂肪とタンパク質のパーセンテージ以外は、DYD又はDRPとDYD又はDRPの遺伝率(h²)の残差共分散は全て遺伝的評価に用いた遺伝率と等しくないという、二つの仮定が立てられた（表２）。
モデル中にPit-1パターンが存在するために、種牛による分散が減少しないので、該方法は、相加的遺伝率（additive heritability）を過大評価する傾向にあるが、この過大評価は、さほど重要ではないはずである。

パターン解間の差として、線形コントラスト（linear contrast）を構築した。コントラストのテストは、以下の統計を用いて行った。
F=b’l-l’C^bbl)^-1l’b
ここでl’bはパターン解間の差を表し、Ｉは線形コントラストベクトルであり、C^bbはパターン効果に関する係数行列の一般化された逆行列のブロックの推定であり、(l’^bbl)^-1は線形コントラストの標準誤差の二乗の逆数であり、分子の自由度は階数(I)=1を用いて近似した。分母は、n-階数(X)=86とした（ここで、nは観察数である）。
あるパターンの存在が唯一つの主要な効果のみを有するかどうかは確実ではない。それ故、該仮定をテストするために、Wellerらの、J. Dairy Sci. 73:2525(1990)に基づく以下の戦略を用いた。
1.パターン間に単一形質の有意なコントラストを示す形質をグルーピングし、最終的な関連形質も含めた。
2.これらの形質間の重み付けした相関Ｖ及び共分散Ｐの行列を得た。
3.カノニカル変換はV=QEQ’（ここでＥは固有値の対角行列、及びＱは固有ベクトルの行列）として定義した。
4.変換行列（transformation matrix）Ｔは、Q^-1S（ここでＳは、元の形質の標準偏差の逆数の対角行列、それ故TPT’＝Eである）
5.変換行列は、関連形質を非関連カノニカル形質に変換するために用いた。
6.カノニカル形質のおよその遺伝率及び重みは、最初の形質に対する値の重み付けした平均として得、重み付け係数は、Q^-1の平方値であった。
7.カノニカル形質は、最初の形質について、上述の方法を用いて分析した。低い相対固有値を示すカノニカル形質は、観察された分散が小さいことを意味する。
8.元の効果に対する多形質線形コントラストは、有意なカノニカルコントラストの逆変換（back transformation）を用いて算定することができる。
9.これらの新規形質に対する結果は、Pit-1パターンの効果が唯一つ観察され得るか、一より多い有意な効果が存在するかどうかを決定するのに有用である。逆変換コントラストは、カノニカル形質に対するPit-1の影響に基づく元の形質間の有意な相違を反映している。
３.結果
PCR/RFLP
PCR産物は、451bp長であった。HinfIによるPCR産物の消化によって、二つの対立遺伝子が明らかとなった。対立遺伝子ＡはHinfIで消化されず、451bp断片を産出し、Ｂ対立遺伝子は一つの制限部位で切断され、上記のWoollardによって記載されたように、長さ244及び207bpの二つの断片を生成する（図１）。
PCR/RFLPとミルク産生の関係
三つのパターンAA、AB、及びBBの頻度は、2.2%、31.5%、及び66.3%であった。Ａ対立遺伝子とＢ対立遺伝子の頻度は、最大確率アプローチ（maximum likelihood approach）によって、、Ａが18.8%、Ｂが81.2%であると推定された。
表３は、３つのPit-1パターン間の線形コントラスト及び標準誤差を示す。それ故、後脚に観察された高度に有意なコントラスト（P<0.01）は、真に生物的な理由よりも、タイプAA動物がこの形質について極端であるという事実によるのかもしれない。ABとBBパターン間の高度に有意なコントラストが、ミルクとタンパク質収量（P<0.01）について見出された。有意なコントラストが、脂肪のパーセンテージと痩身度（P<0.05）に観察された。ABパターン又はAAパターンは、ミルク、タンパク質収量、及び痩身度が高く、脂肪パーセンテージが低かった。これらの結果は、ミルク産生に対するヘテロ接合体AB又はAAの単一の正の作用から生じるものと解釈することができ、それによりタンパク質収量に正の影響を及ぼし、脂肪収量には及ぼさず、脂肪パーセンテージに観察された負の影響を与える。該線形形質（linear trait）は、ミルク収量と強く関連しているので、痩身度に対するPit-1の影響は、この意味で、さして驚くべきものではない。

単一作用の仮定をテストするために、我々は、ミルク、脂肪とタンパク質収量のカノニカル変換を行った。パーセンテージDYDが収量の関数として得られたので、収量を分析し、それ故、この結果は新しい情報を全くもたらさない。痩身度を加えた。表現型相関行列を算出した。観察結果は、有効娘数を用いて重み付けした。これらの数は、収量及びタイプ形質について異なっていたので、重み付けした有効娘数の平均としておよその重量を得た。表４は、相関を示している。収量形質間の相関は、及び脂肪と他の形質の一つとの間の相関に比べて、高い相関を有する期待値を示した。痩身度は、収量形質と0.42〜0.51の相関を示した。

相関行列のカノニカル分析の結果が、表５に示されている。第一及び第二のカノニカル形質が、全分散の90%を説明する。特に、最後のカノニカル形質は、あまり情報を与えなかった。表５は、固有ベクトル及び各固有ベクトル中の様々な形質の相対的な重要性も示している。第一のカノニカル形質は、痩身度について15%、タンパク質について30%の間の相対的な影響を有する４つの形質を全て組み合わせたものである。しかし、第二のカノニカル形質は、該形質に81%の相対的重要性を有する痩身度と、より特異的に連関している。第三のカノニカル形質は、脂肪と関連し、ミルクと関連はこれより弱く、第四のカノニカル形質は、ミルクとタンパク質のみと関連している。

表６は、四つのカノニカル形質にみられた線形コントラスト及び標準誤差を示している。予期に反して、AB及びBBパターンとのコントラストに対して、第一及び第二のカノニカル形質は極めて有意であり（P<0.001）、第四のカノニカル形質は若干有意であった（P<0.05）。この結果は、Pit-1が一より多くの作用を有し得ることを示していた。第一のカノニカル形質は、痩身度に対してより特異的に連関する。最後の形質は、ミルクとタンパク質収量間の平衡を反映していた。これらのコントラストをよりよく理解できるように、表7には、元のスケールで表したコントラストの値と標準誤差を掲げてある。我々は、第一のカノニカルコントラストのために、逆変換したコントラストがミルク、脂肪とタンパク質に極めて重要であることを観察した。AB動物では全てが正であり、BB動物より勝っていた。第二のカノニカル形質については、ABがミルク、脂肪とタンパク質で劣っており、痩身度では勝っていた。このこともまた、第一に収量と痩身度の関連を通じて、さらには収量に対して僅かに負の影響を与えながら直接痩身度に影響を与えることによって、痩身度に対するPit-1の影響が重要であるらしいということを示す。三番目のカノニカル形質は有意なコントラストを示さず、四番目のカノニカル形質は、有意ではあったが、分散全体のごく僅かしか説明しなかった。有意なカノニカル形質を全て集めると、単一形質の場合のように、より高い群を成すコントラストが観察された。これは、脂肪収量及び痩身度の場合に特に明確であったが、ミルク及びタンパク質についても明確であった。複数形質コントラストには、相関した形質、特に脂肪及び痩身度からの情報を含むことがその理由と思われ、このことは該相違を説明し得るかもしれない。コントラストの標準誤差は、さほど増加せず、ミルクと脂肪収量についてはむしろ減少していた。

例Ｂ
Pit-1遺伝子の配列決定及び突然変異の特定
該方法は、定まった点から始まり、決まった残基又は残基の組み合わせでランダムに終わる放射線ラベルされたオリゴヌクレオチドの独立した群を生成する。DNA中の全ての塩基は、可変的な末端となる機会が等しいので、各群は、元のDNAの長さに沿ったある塩基の位置によって長さが決まる、オリゴヌクレオチドの混合物からなる。これらのオリゴヌクレオチド群は、一ヌクレオチドしか長さが違わない各DNAが識別され得る条件下で電気泳動を行うことによって分離される。配列決定ゲルの隣接したレーン中に該群をロードすると、ゲルのオートラジオグラフィー像から直接、DNA中のヌクレオチドの順番を読み取ることができる。
参照文献：Sanger, F., S. Nicklen, ＆ A.R. Coulson 1977,チェーン−ターミネーション阻害剤によるDNA配列決定、Proc. Natl. Acad. Sci. 74:5463
例Ｃ
プライマーを用いた検出実験
1)リガーゼ連鎖反応
オリゴヌクレオチドプローブとDNAリガーゼのみを利用するリガーゼ連鎖反応（LCR；ligase chain reaction）は、大過剰の他のDNA配列情報の存在下で特定の標的DNA配列の約1000コピーを検出することができる。1989年に最初に記載されて以来(Backman ＆ Wang, 1989,欧州特許出願第0,320,308号；Royerら、1989、欧州特許出願第0,324,616号；Wallace, 1989,欧州特許出願第0,336,731号；Wu＆Wallace, 1989, Genomics 4:560-569；Orgel, 1989；Richards＆Jones, 1989),非放射性検出とともに熱安定性DNAリガーゼを利用することによって、LCRは改良されてきた（Bondら、1990）。

2)HinfI制限酵素を用いるPit-1遺伝子のFLPは、上記Woolardらに適合させたPCR分析によって明らかとなった。
上記のSangerらによって記載された方法を用いて、我々は、記録した（reproted）RFLPに付随する1178位のヌクレオチドの点変異（aからg）を同定した。また、HinfI制限酵素を用いず、該突然変異をカバーするプライマーを用いる新規PCR法も開発した。
ポリメラーゼ連鎖反応法
Pit-1遺伝子のRFLPは、PCRによって明らかとなった。簡潔に述べれば、第三のプライマー（プライマーＢ＝5’-GACAGGGAAAGTGATATAGAAAGGGAGATAGA-3’）とともに、突然遺伝子を内包する二つのPCRプライマー（プライマーAA＝5’-CAGAGAGAAAAACGGGTGAAGACAAGCATA-3’及びプライマーBB＝5’-CAGAGAGAAAAACGGGTGAAGACAAGCATG-3’）を用いて、2mM MgCl₂を含有する50μＬの反応量のゲノムDNAから360bp断片を増幅した。条件は、95℃で３分の後、95℃で１分、65.2℃で１分、及び72℃で１分を35サイクルであった。最終ステップは、72℃10分であった。１μg/mLの臭化エチジウムを用いて、2%のアガロースゲル上でPCR産物を電気泳動した（図３。）
結論
HinfIにによって認識される制限部位を用いて、Pit-1遺伝子（プロラクチン及びGH遺伝子発現を活性化するのに必要な成長ホルモン因子-1／下垂体特異的転写因子）では二つの対立遺伝子が区別された。消化されないＡ、及び該部位を示すＢという二つの対立遺伝子が観察された。AAパターンは、AB又はBBパターンに比べて頻度が小さい。ミルク、タンパク質、及び痩身度は、Pit-1AB又はAAパターンが、BBに比べて有意に勝っていることが観察された。このことは、ヘテロ接合の動物の方が、生産性が高く、よりよい乳牛であることを示している。脂肪のパーセンテージは、BB動物よりもABの方が低く、ほぼ一定の脂肪収量で、より多くのミルクが産生された結果である。
これらの結果は、Pit-1の一つの作用を示している。しかし、カノニカル変換アプローチを用いることによって、Pit-1の少なくとも二つの作用が観察された。一つは収量及び痩身度に対してであり、もう一つは痩身度だけに対してである。これらの結果は、プロラクチン及びGH遺伝子発現の活性化を通じて、Pit-1が一以上の役割を有していることを説明できる。第一の役割は、ミルク、タンパク質（及び脂肪）収量であり、第二の役割は、動物の筋肉の成長に関連し、ABの存在によって、痩身度が増大して筋肉性が減少することを意味している。
非常に興味深いことに、これらの発見は、関連形質に対する作用を識別するのに、カノニカル変換が有用であることを示している。オリジナルに対してPit-1多型と乳牛のミルク形質との関連が示されたが、変換されたスケールに対しても示された。痩身度を除くコンフォメーション形質（ミルク収量と関係する形質）では、関係の重要性は低かった。また、カノニカル変換は、痩身度に対する効果は、その一部が、ミルク形質に対するPit-1の直接的な影響の結果によるものにすぎないことを示した。
さらに、Pit-1遺伝子中の特異的な突然変異の同定は、様々な対立遺伝子と対応する形質とを識別するために実施する迅速且つ高感度の方法を可能にする。Background of the Invention
Field of Invention
The present invention relates to genetic markers associated with various conformational traits. More specifically, the present invention describes a method that uses polymorphisms of the Pit-1 gene to readily determine animal traits such as milk production and muscularity.
Description of prior art
Currently, it takes a great deal of cost and time to select certain traits in mammals. Typically, the screening process involves a phylogenetic assessment of the long-term mammalian history. This assessment is based on various mammalian or animal traits such as weight at birth, body weight at growth, build, strength, firmness, marbling, color, and so on.
To date, many selections of certain traits in animals involve visually identifying specific traits within a time frame or measuring the weight of an animal at a certain point in time. Subsequently, animals with the quality trait to be screened are crossed with similar animals so that the trait is expected to be dominant in the next or subsequent generations.
Current methods for character selection in mammals are often laborious and must wait for the judgment of a specialist in the field, such as a breeder. However, the screening performed is not necessarily dominant in the next generation and beyond. For example, to screen for dairy cows that are good milk-producing animals, such screening takes 36-48 months, and after screening is done, it is often based on assumptions and the judgment of the breeder.
In view of the uncertainties, costs, and time associated with current animal trait sorting methods, new methods are currently being developed that utilize more scientific processes that would improve the sorting process.
One such method involves the study of candidate genes to determine whether a specific gene is associated with an animal's conformational trait, and therefore these genes screen for a desired trait. Can be used as a molecular marker. The method first requires identification of a candidate gene or an unknown genetic marker associated with the desired trait. The candidate gene approach may work, but the first gene must be identified in the desired species and it must be correlated to the desired trait.
The somatotropin system has several genes that may play a role in regulating certain traits in animals because they are related to growth, lactation, reproduction, and immunity. The somatotropin system is extremely complex, somatoclinin and somatostatin at the hypothalamic level; pituitary-specific transcription factor (Pit-1) required for growth hormone expression in mammals at the pituitary level At the liver level, growth hormone receptor and growth hormone cytoplasmic transport protein; at the cellular level, growth hormone receptor, insulin growth factor-1 and insulin growth factor transport protein are included.
Since the system has different functions in different parts of many animals, from the pituitary to the cellular level, it is very complex to select genes from the somatotropin system that can affect certain traits in animals. is there.
The present invention relates to selection of a pituitary-specific transcription factor (hereinafter referred to as Pit-1) gene that can act as a genetic marker for specifying a specific trait of an animal.
Pit-1 is a member of the POU family of homeodomain transcription factors and plays an important role in the growth process. Originally, the POU domain was the product of three mammalian transcription factors, Pit-1, Oct-1, Oct-2, and the nematode gene unc-86 (Herr et al., Genes & Dev. 2: 1513 (1988); Ruvkun And Finnery, Cell 64: 475 (1991)), identified as a highly conserved region of 150-160 amino acids.
Pit-1 is a pituitary-specific transcription factor that regulates growth hormone and activates prolactin and has a role in pituitary cell differentiation and proliferation (Steinfelder et al., PNAS, USA 88: 3130 (1991)). . Mutations in the Pit-1 gene cause a dwarf phenotype in Snell & Jackson mice, resulting in prehypophyseal hypoplasia (Li et al., Nature 347: 528 (1992)). Furthermore, inhibition of Pit-1 synthesis has been shown to cause a decrease in prolactin and growth hormone (GH) expression and a dramatic decrease in cell proliferation in GH and prolactin producing cell lines (McCormick et al., Nature 345). : 829 (1990)).
In humans, patients with familial pituitary hypoplasia (Pfaffle et al., Science 257: 1118 (1992)); patients with sporadic complex pituitary hormone deficiency (Radovick et al., Science 257: 1115 (1992); Tatusmi et al., Nature Genetics 1:56 (1992)) also reported various mutations in the Pit-1 gene.
The cDNA sequence of bovine Pit-1 was published by Bodner, M. et al., Cell 55 (3): 505-568 (1988) and is shown in FIG.
The association of pig growth and death traits with the Pit-1 polymorphism has been described by Yu et al., J. Anim. Sci. 73: 1282 (1995). Yu et al. Described 3 Pit in pigs based on PCR / RFLP using two restriction fragment length polymorphisms (hereinafter referred to as RFLP), RsaI using Pit-1 POU domain cDNA probe and restriction enzymes BamHI and MspI. -1 polymorphism was described.
From the results of the above mixed model analysis by Yu et al., It became clear that pigs with MspI CC genotype had a higher birth rate than pigs with DD genotype. Furthermore, in the presence of the Pam-1 BamHI polymorphism, high birth rates were significantly associated with BB genotypes, but the authors should not conclude such associations because the BB genotype population is very small We are careful.
Woolard et al., J. Anim. Sci. 72: 3267 1994) found a Hinfl polymorphism at the bovine Pit-1 locus, but these authors found that the mutation was associated with the animal trait I couldn't. The conclusion described by Woolard et al. Above was that the observed polymorphic fragments were consistent with autosomal Mendelian inheritance.
The disclosures of Yu et al. Or Woolard et al. Disclose no association between allelic pattern AB and milk production, nor between allelic pattern BB and animal muscularity.
The present invention therefore overcomes the shortcomings of current methods in animal trait sorting by providing a scientific basis for trait sorting through the use of genetic markers.
Furthermore, the method described in the present invention can be used to identify superior milk producing animals from animals having meat producing characteristics.
A surprising discovery has been made that polymorphisms in the Pit-1 gene can be used to identify traits such as animal milk production and muscularity. For the Pit-1 gene required for activation of prolactin and growth hormone gene expression, two alleles A and B were distinguished. The AA pattern was less frequent than the AB or BB pattern. It was observed that the Pit-1 AB pattern or AA pattern was significantly better than the BB pattern for milk, protein, and angularity. Similarly, BB genotype patterns were associated with animal muscularity.
This discovery allows the use of mutations in the Pit-1 gene as genetic markers to identify certain traits in animals. Once these traits have been identified, because of their superior traits, the animals can sell high on the market.
Accordingly, it is an object of the present invention to provide genetic markers for character selection in animals.
In another aspect, the present invention provides a method for identifying animals with superior milk production or muscle traits.
In yet another aspect, the present invention provides genetically engineered animals with superior milk production, leanness, fat, protein, or muscle traits. These and other objects are achieved by the present invention as defined by the summary of the invention, the description of the preferred embodiments, and the claims.
Summary of the Invention
The present invention thus provides genetic markers that can be used for mammalian trait selection.
Furthermore, the present invention is a method for identifying a polymorphism present in the Pit-1 gene, wherein the polymorphism selects an animal's excellent leanness, fat, muscle, protein, or milk production trait. Provide a method that can be used to:
Thus, in one aspect of the composition, the present invention is a genetic marker used to identify a trait for milk production or meat production from an animal, the genetic marker comprising: The present invention relates to a genetic marker having mutations in a fragment of the Pit-1 gene, in which three allelic patterns are observed, and the pattern of complete mutation is used as an index.
In the present application, the marker characteristic of milk production ability is represented as AA for the homozygous allele, and the marker characteristic of meat production ability is represented as BB for the homozygous state.
As demonstrated by the inventors in the experiment shown in Example B, the sequences of alleles A and B are the Pit-1 AA adenosine present at position 1178 of the sequence of FIG. The only difference is that it is translocated to guanine.
In a preferred embodiment of the invention, three allelic patterns are identified and digested completely after digestion with a restriction endonuclease that cleaves a mutated Pit-1 gene fragment but not a non-mutated Pit-1 gene fragment. The pattern is an indicator of the trait for the animal's muscularity, while a moderately digested / non-digested pattern or a complete non-digested pattern is an indicator of the animal's milk production trait.
In a more preferred embodiment of the invention, the restriction endonuclease used is HinfI.
In another preferred embodiment of the present invention, three allelic patterns are identified using a probe covering the mutated region in the Pit-1 gene, and one probe is specific to the mutated Pit-1 gene. Another probe is specific for the non-mutated Pit-1 gene.
In another aspect, the invention relates to a method for detecting an animal trait, wherein the process comprises:
(1) isolating genomic DNA from animals;
(2) isolating a fragment as needed from the genomic DNA comprising the Pit-1 gene fragment;
(3) detecting a mutation in the Pit-1 gene;
(4) In order to determine the traits of the animal, the mutation is analyzed, and in the analysis, the muscle and fat traits of the animal can be distinguished from milk production traits.
It is related with the method which comprises these steps.
In certain embodiments of the invention, detection is achieved by using a restriction endonuclease.
In another embodiment of the invention, detection is achieved by using a mutated gene in the Pit-1 gene, more specifically a probe covering position 1178.
In yet another aspect, the present invention relates to genetically engineered animals having the characteristic traits described in the present invention.
Brief description of the drawings
FIG. 1 is an electrophoresis gel showing a PCR / RFLP pattern using the restriction enzyme HinfI for the Pit-1 gene, observed in Holstein-Friesian and Simmental cattle. The left is the size of the digested fragment and the top is the pattern. The fragment length (kilobase) was estimated by comparison with the DNA size marker φX174DNA / HaeIII fragment.
FIG. 2 is the sequence of bovine Pit-1 cDNA.
FIG. 3 is an electrophoresis pattern showing a PCR amplification product obtained after amplification with the following primers.
1-3-5 rows: Pit1 AA and Pit 1B
Row 2-4-6: Pit1 BB and Pit 1B
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
As used herein, “animal” includes, but is not limited to, all mammals, birds, fish, including dairy cows, bulls, goats, pigs, sheep, chickens, and the like. Due to the high conservation (> 95%) of the Pit-1 gene between species, the present invention can be easily transferred from one species to another. The invention can also be used to determine traits in humans such as the ability to metabolize growth hormone.
The term “polymorphism” refers to the simultaneous occurrence of genomes within a population that exhibit allelic variations, such as those found in either alleles that lead to different phenotypes or changes in DNA that affect restriction patterns means.
As used herein, the term “trait” includes any characteristic, but specifically indicates what distinguishes one animal from another.
As used herein, the term “angularity” refers to an objective criterion used to identify a specific trait of an animal with respect to specific measurements that can be made on the animal's body. The measurement is made for certain morphological characteristics of the animal.
For example, to determine the leanness for a milk production trait, the animal's pelvic bone and the muscles surrounding the pelvic bone are measured to determine if they are protruding. Next, the scale can be determined. When the bones are very protruding, there is almost no muscle around and therefore the animal is milk producing. Conversely, when the bone is not protruding, the animal has a lot of surrounding muscle, and it is considered that the animal is not a good milk-producing cow but rather a meat-producing cow.
In the context of the present invention, the term “muscularity” includes animals suitable for meat-producing cows that can be slaughtered to obtain meat rather than milk-producing cows.
More specifically, the present invention relates to the use of the Pit-1 gene polymorphism as a potential marker for genetic variation in animals. Pit-1 encodes a transcription factor in the cell, and any mutation in the gene can alter transcriptional capacity by attenuating or enhancing, thereby revealing various traits in animals. A polymorphic form is brought about.
The Pit-1 gene was previously identified in the 13-kb bovine genomic library by Woolard et al. The 13-kb clone was isolated from the library by using bovine Pit-1 cDNA as a probe (labeled).

The clone was identified as a bovine Pit-1 genomic fragment by characterization and sequencing of the XhoI, HinfI, and EcoRI subclones of the 13-kb insert by restriction enzyme digestion.
Similarly, the methods taught by Woolard et al. Above can be used to identify the Pit-1 gene in various genomic libraries other than cattle. This will allow the identification of specific sequences within the Pit-1 genomic fragment that can be used to amplify the sequences from various animals, as described below.
The first step in identifying a mutation in the animal's Pit-1 gene is to obtain a sample (including but not limited to) semen, blood, cells, biopsy tissue, feces, etc. from the animal. Next, genomic DNA can be extracted from samples obtained using methods known in the art, such as described in Sambrook et al., “Molecular Cloning, A Laboratory Manual, second edition, 1989”.
However, genomic DNA is extracted using the procedure described in Walsh's Biotechniques, 10: 506 (1991) for semen and the procedure described in Lewin and Stewart-Haynes Biotechniques, 13: 522 for blood. It is preferable to do.
After extracting genomic DNA, there are several known methods in the art for detecting mutations in the Pit-1 gene. Any detection method can be utilized to detect the mutation. Examples of these methods include RFLP, SSCP, DGGE, CFLP, and single nucleotide mutations as described by Prosser, Trends Biotech 11: 238-246 (1993), and Sambrook et al. It is not limited to. These methods are discussed in further detail below.
For example, in the RFLP method, PCR primers are used to amplify a fragment containing the Pit-1 gene by standard procedures. Any PCR primer that allows amplification of the Pit-1 sequence can be utilized, and methods of isolating specific clones that identify such primers can be utilized.
In a preferred embodiment of the present invention, PCR primers can be designed from intron V and exon 6 of a fragment containing a polymorphism of the Pit-1 gene, such as the 451-bp fragment described by Woolard et al. . In a further preferred embodiment of the invention, the PCR primers are as follows:

Amplification of the Pit-1 fragment can be performed using standard PCR procedures such as those described in Sambrook et al. However, 2 mM MgCl₂Preferably, genomic DNA is amplified in a 50 μL reaction volume containing.
In a preferred embodiment of the present invention, at 88-98 ° C for 10-15 minutes, at 90-100 ° C for about 1 minute, then 90-50 ° C for 20-40 seconds for 25-50 cycles; PCR reaction conditions of 5 minutes; 68-80 ° C. for about 1-5 minutes can be used. The final step may include a cycle of 8-12 minutes at 68-80 ° C.
Following amplification, the specific mutation in Pit-1 is subsequently cleaved using various restriction enzymes or endonucleases known in the art. These restriction enzymes include, but are not limited to, BamHI, EcoRI, SmaI, HinfI and the like. See, for example, the enzymes described in Samrook et al. Of particular interest is the use of a restriction endonuclease that cleaves the mutated allele of the Pit-1 gene and does not cleave the unmutated allele of the Pit-1 gene. In a preferred embodiment of the invention, HinfI is used for the identification of animal milk production, fat, protein, and muscle traits.
Following digestion, the sample is then electrophoresed on an agarose gel and identified using a stain such as ethidium bromide (but any stain that identifies fragments can be used).
SCCP (single-stranded DNA conformation polymorphism) is also a method known in the art that can identify one or more mutations in an isolated genomic Pit-1 fragment. is there. The method is based on PCR amplification using primers similar to those described above. Next, the amplified fragment³²Label with a label such as P or any other suitable radioactive label. Next, the radioactively labeled fragments are denatured, for example, by rapid cooling after heating. After cooling, the fragments are electrophoresed using non-denaturing techniques and then subjected to audioradiographed.
DGGE (denaturing gradient gel electrophoresis) is yet another method for detecting Pit-1 mutations. In the method, the fragments are amplified by PCR using appropriate primers as described above and subjected to a denaturing gradient. In addition, the sample is electrophoresed to detect mutations.
Yet another method that can be used to detect mutations is CFLP (cleavage fragment length polymorphism). The method can detect that a single base is mutated in the DNA sequence between two molecules, wild type DNA and mutant DNA. The method is currently marketed by Boehringer Mannheim and can be purchased in kit form.
Another method that can be used to detect mutations in the Pit-1 gene utilizes primers that cover the mutated region of the Pit-1 gene.
More preferably, two sets of primers (a pair that covers and contains a mutated Pit-1 gene, a primer that covers a non-mutated Pit-1 gene and is specific for this) Two separate amplification reactions are performed on the extracted genomic DNA sample. More preferably, the primer used is labeled so that the amplification product can be easily visualized. According to the method, when the tested genomic DNA contains a homozygous mutant Pit-1 gene (two mutated alleles), only the amplification reaction using a probe specific for the mutated region signals. (Ie, an amplification product) will be produced. When the tested genomic DNA is heterozygous (ie, one mutated allele, one non-mutant allele), two proliferative reactions will produce a signal (amplified product). Similarly, when the tested genomic DNA contains a homozygous non-mutated Pit-1 gene, only an amplification reaction using a probe specific for the non-mutated region will produce a signal. Therefore, a single amplification step reveals the allelic pattern of the tested DNA.
In another aspect, the use of the technique described in WO 97/06276 is particularly suitable for detecting the homozygous or heterozygous state of a marker in a single step.
In these methods, it is not necessary to further cleave the amplified product in order to distinguish among the various patterns. Furthermore, it is not necessary to amplify a specific Pit-1 gene fragment prior to the detection step. Finally, depending on the nature of the label used, visualization can be very easy. For example, if the probe is radiolabeled, the probe is obtained by electrophoresis. More interestingly, direct visualization is obtained when the probe is labeled with a stain.
In the first embodiment, the test can be performed in a very simple device such as a plate. A sample of genomic DNA in two wells (one with a set of labeled probes that cover a mutation in the Pit-1 gene and contain one probe specific for the mutated Pit-1 gene) And the other contains a set of probes that cover a Pit-1 gene mutation and contain one probe specific for the non-mutated Pit-1 gene).
After amplification, the label appears directly in the plate and can be analyzed by automated equipment.
In the amplification method, the second primer used in each set of probes is selected to allow amplification of products containing 200-400 bp, more preferably 320-370 bp. Such a second primer can be selected from the following primers, for example.

The length of each primer preferably comprises 20-40 bases, more preferably 25-35.
Selection of an appropriate probe for the strategy is enabled by the inventors identifying the mutation in the Pit-1 gene that is responsible for the observed polymorphism. More specifically, the mutation occurs at nucleotide 1178 in the Pit-1 coding region, with adenine replaced by guanine in the mutated gene. The mutation is shown in FIG.
The position of the probe covering the mutated region (mutation) can vary.
More preferably, in the first aspect, the two sets of primers are for the AA genotype characteristic of milk production ability:

About BB genotype that is characteristic of meat production ability,

It is.
In another embodiment, when using WO 97/06276,
Two sets of primers that produce amplified fragments of various sizes are:
For the AA genotypes that characterize the milk-producing brain,

The second primer Pit-1 C ′ has a BB genotype characteristic of meat producing brain that is at least 10 bp shorter or longer than that obtained using Pit-1 AA and Pit-1B. Choose to be amplified.
Although many mutation detection methods are available, the present invention is not limited to the methods discussed above, and includes all methods of detecting mutations.
Subsequently, alleles and allele patterns are identified, and then statistical analysis is performed to determine specific traits that are revealed by identifying alleles. More specifically, any statistical program that can identify daughter yield deviation (DYD) and deregressed proof (DRP) may be utilized. Statistical analysis is preferably performed using SAS MIXED operations (User Guide, Statistics, Version 6, 4th ed. SAS Inst., Inc. Cary, N.C. (1990)). Technical Report P 229 SAS Inst., Inc., Cary, N.C. (1992). The statistical analysis used in the present invention is described in detail in the following example.
The present invention also includes kits containing materials necessary for visualizing mutations such as genomic DNA extraction materials, PCR primers having SEQ ID NOs: 1 and 2 (described above), and electrophoresis gels. The contents of the kit may vary depending on the detection method used, as described in detail above.
Primers covering mutations in the Pit-1 gene are also included in the present invention.
More specifically, the present invention also relates to a primer comprising 20-40 bases complementary to a region of the Pit-1 gene having a single mutation.
The present invention also includes a set of primers that allows amplification of a 200-400 base region in the Pit-1 gene, which region contains a single mutation.
The following primers are included in the present invention.

In order to further illustrate the present invention and its advantages, the following specific examples are given, but it should be understood that they are merely illustrative and not limiting in any way.
Example A
1. DNA extraction and PCR
Genomic DNA of 89 commercially available Italian Holstein-Friesian cattle was extracted from semen as described by Lucy et al., Domest. Anim. Endocrinol. 10: 325 (1993).
RFLP of Pit-1 gene using HinfI restriction enzyme was revealed by PCR analysis adapted to the above-mentioned Woolard et al.
PCR primers were designed from intron V and exon 6. The sequences of the primers used were 5'-AAACCATCATCTCCCTTCTT-3 '(SEQ ID NO: 1) and 5'-AATGTACAATGTGCCTTCTGAG-3' (SEQ ID NO: 2). These primers are 2 mM MgCl2₂Was used to amplify a 451 bp fragment from genomic DNA in a 50 μL reaction volume. The conditions were 35 cycles of 94.5 ° C. for 10 minutes, 94 ° C. for 1 minute, 95 ° C. for 30 seconds, 56 ° C. for 1 minute, and 72 ° C. for 2 minutes. The last step was 72 ° C for 10 minutes. The PCR product was digested with HinfI and electrophoresed on a 2% agarose gel using 1 μg / mL ethidium bromide (FIG. 1).
Daughter yield deviations (DYD) calculated in March 1996 were obtained from Holstein-Friesian cattle obtained from the Italian Holstein-Friesian Breeder Association ANAFI (Associazione Nazionale Allevatori Frison Italiana, Cremona, Italy). DYD values are not calculated for fat and protein percentages. These traits can only be indirectly estimated from solutions for yield traits and average population values for these traits. Therefore, the DYD value was calculated using the same approach as the method for calculating the genetic value for the percentage trait.
Similar DYDs were not available for type trait. Therefore, the gene value was converted to a non-regression proof (Banos et al., Interbull Annual Meeting, Aarhus, Denmark, Bulletin No. 8, 1993, sigbjorn et al., J. Dairy Sci, 78: 2047 (1995)) Can be considered to approximate DYD.
Table 1 shows the mean and standard deviation of DYD and / or conformational trait DRP for milk production traits in bovine samples. An effective number of daughters (measured number of daughters in the group adjusted for distribution) was obtained for the yield trait, but not for the type trait. Therefore, the following formula:
"Effective number" = "true number" x "square root of the ratio of the number of groups to the number of daughters"
Was approximated using

2. Statistical analysis
Statistical analysis was performed using the SAS MIXED operation. The mixed model used is
y = Xb + Zu + e
Where y = vector of bovine DYD or DRP; b = vector of fixed effect associated with the Pit-1 pattern; u = vector of bovine random additive polygene effect, and e = other random effects It was a vector. The model was solved using the following mixed model equation:

Where A is an additive relationship matrix (89 known ancestors 1842) between 89 cattle constructed using known whole-kinship relationships, R^-1= D / δ² _eHere, D is a diagonal matrix of the number of effective daughters for all cows present on the diagonal. Next, the residual variance δ² _eThe matrix is divided by the estimated value. This is an estimated REML (Patterson and Thompson, Biometrika 58: 545 91971), which converges in one round, so this is a non-interactive minimum variance quadratic estimate (non-interactive minimum variance quadratic) unbiased estimation) (Rao, J. Mult. Anal. 1: 445 (1971)). The resulting estimate has a secondary form characteristic that minimizes the variance of the sampling. Heritability of DYD or DRP and DYD or DRP, except for the percentage of fat and protein assumed to be heritability of 0.50 (h²Two assumptions were made that the residual covariance of) was not equal to the heritability used in the genetic evaluation (Table 2).
The method tends to overestimate the additive heritability because the Pit-1 pattern in the model does not reduce the dispersion due to the cattle, but this overestimation is so important Should not.

As a difference between pattern solutions, a linear contrast was constructed. The contrast test was performed using the following statistics.
F = b’l-l’C^bbl)^-1l’ b
Where l′ b represents the difference between the pattern solutions, I is the linear contrast vector, and C^bbIs an estimate of the block of the generalized inverse matrix of the coefficient matrix for the pattern effect, and (l ′^bbl)^-1Is the reciprocal of the square of the standard error of linear contrast, and the degree of freedom of the numerator was approximated using rank (I) = 1. The denominator was n-order (X) = 86 (where n is the number of observations).
It is not certain whether the existence of a pattern has only one major effect. Therefore, the following strategy based on Weller et al., J. Dairy Sci. 73: 2525 (1990) was used to test the assumption.
1. Traits showing significant contrast of a single trait between patterns were grouped and the final associated trait was included.
2. A matrix of weighted correlation V and covariance P between these traits was obtained.
3. The canonical transformation was defined as V = QEQ '(where E is a diagonal matrix of eigenvalues and Q is a matrix of eigenvectors).
4. The transformation matrix T is Q^-1S (where S is the diagonal matrix of the reciprocal of the standard deviation of the original trait, hence TPT '= E)
5. The conversion matrix was used to convert related traits to unrelated canonical traits.
6. The approximate heritability and weight of the canonical trait are obtained as a weighted average of the values for the first trait and the weighting factor is Q^-1Was the square value.
7. Canonical traits were analyzed for the first trait using the method described above. A canonical trait that exhibits a low relative eigenvalue means that the observed variance is small.
8. The multi-trait linear contrast to the original effect can be calculated using a significant canonical contrast back transformation.
9. The results for these new traits are useful in determining if only one effect of the Pit-1 pattern can be observed or if there is more than one significant effect. The inverse transform contrast reflects a significant difference between the original traits based on the effect of Pit-1 on the canonical traits.
3. Results
PCR / RFLP
The PCR product was 451 bp long. Digestion of the PCR product with HinfI revealed two alleles. Allele A is not digested with HinfI, yielding a 451 bp fragment, and the B allele is cut at one restriction site, producing two fragments 244 and 207 bp in length, as described by Woollard above. (FIG. 1).
Relationship between PCR / RFLP and milk production
The frequencies of the three patterns AA, AB, and BB were 2.2%, 31.5%, and 66.3%. The frequency of the A and B alleles was estimated to be 18.8% for A and 81.2% for B by the maximum likelihood approach.
Table 3 shows the linear contrast and standard error between the three Pit-1 patterns. Therefore, the highly significant contrast (P <0.01) observed in the hind limb may be due to the fact that type AA animals are extreme about this trait rather than truly biological reasons. A highly significant contrast between AB and BB patterns was found for milk and protein yield (P <0.01). Significant contrast was observed in fat percentage and leanness (P <0.05). The AB pattern or AA pattern had higher milk, protein yield, and leanness, and a lower fat percentage. These results can be interpreted as arising from a single positive effect of heterozygote AB or AA on milk production, thereby positively affecting protein yield, not fat yield, fat Observe negative effects on percentages. Since the linear trait is strongly associated with milk yield, the effect of Pit-1 on leanness is not surprising in this sense.

To test the single action hypothesis, we performed a canonical transformation of milk, fat and protein yield. Since the percentage DYD was obtained as a function of yield, the yield was analyzed and therefore this result does not give any new information. Added leanness. A phenotypic correlation matrix was calculated. The observation results were weighted using the number of effective daughters. Since these numbers differed for yield and type traits, an approximate weight was obtained as an average of the weighted effective daughter numbers. Table 4 shows the correlation. The correlation between the yield traits showed an expected value with a high correlation compared to the correlation between fat and one of the other traits. The leanness showed a correlation between yield traits and 0.42-0.51.

The results of the canonical analysis of the correlation matrix are shown in Table 5. The first and second canonical traits account for 90% of the total variance. In particular, the last canonical trait did not give much information. Table 5 also shows the relative importance of the eigenvectors and the various traits in each eigenvector. The first canonical trait is a combination of all four traits with a relative impact of between 15% for leanness and 30% for protein. However, the second canonical trait is more specifically associated with leanness with 81% relative importance to the trait. The third canonical trait is associated with fat, less associated with milk, and the fourth canonical trait is associated with milk and protein only.

Table 6 shows the linear contrast and standard error observed for the four canonical traits. Unexpectedly, the first and second canonical traits were very significant (P <0.001) and the fourth canonical trait was slightly significant (P <0.05) for contrast with AB and BB patterns. ). This result indicated that Pit-1 can have more than one action. The first canonical trait is more specifically associated with leanness. The last trait reflected an equilibrium between milk and protein yield. To better understand these contrasts, Table 7 lists the contrast values and standard errors in the original scale. We have observed that for the first canonical contrast, the inverse transformed contrast is crucial to milk, fat and protein. In AB animals everything was positive and better than BB animals. Regarding the second canonical trait, AB was inferior in milk, fat and protein, and was superior in leanness. This is also important for the impact of Pit-1 on leanness, primarily through the relationship between yield and leanness, and directly affecting leanness with a slight negative impact on yield. It seems that it seems. The third canonical trait showed no significant contrast, while the fourth canonical trait was significant but accounted for very little of the overall variance. When all significant canonical traits were collected, a higher group of contrasts was observed, as in the case of a single trait. This was particularly clear in the case of fat yield and leanness but also for milk and protein. The reason may be that the multi-trait contrast includes information from correlated traits, especially fat and leanness, which may explain the difference. The standard error of contrast did not increase much, but rather decreased for milk and fat yield.

Example B
Pit-1 gene sequencing and mutation identification
The method produces an independent group of radiolabeled oligonucleotides that start at a fixed point and end randomly with a fixed residue or combination of residues. Since every base in DNA has the same chance of becoming a variable end, each group consists of a mixture of oligonucleotides whose length is determined by the position of a base along the length of the original DNA. These oligonucleotide groups are separated by electrophoresis under conditions that allow each DNA that differs in length by only one nucleotide to be identified. When the group is loaded into adjacent lanes of a sequencing gel, the order of nucleotides in the DNA can be read directly from the autoradiographic image of the gel.
References: Sanger, F., S. Nicklen, & A.R. Coulson 1977, DNA sequencing with chain-termination inhibitors, Proc. Natl. Acad. Sci. 74: 5463
Example C
Detection experiment using primers
1) Ligase chain reaction
Ligase chain reaction (LCR) using only oligonucleotide probes and DNA ligase can detect about 1000 copies of a specific target DNA sequence in the presence of a large excess of other DNA sequence information. Since first described in 1989 (Backman & Wang, 1989, European Patent Application No. 0,320,308; Royer et al., 1989, European Patent Application No. 0,324,616; Wallace, 1989, European Patent Application No. 0,336,731; Wu & Wallace, 1989, Genomics 4: 560-569; Orgel, 1989; Richards & Jones, 1989), LCR has been improved by utilizing thermostable DNA ligase with non-radioactive detection (Bond et al., 1990).

2) FLP of Pit-1 gene using HinfI restriction enzyme was revealed by PCR analysis adapted to Woolard et al.
Using the method described by Sanger et al. Above, we identified a point mutation (a to g) at nucleotide position 1178 associated with the reproted RFLP. We also developed a new PCR method that uses primers that cover the mutation without using the HinfI restriction enzyme.
Polymerase chain reaction method
The RFLP of the Pit-1 gene was revealed by PCR. Briefly, two PCR primers (primer AA = 5′-CAGAGAGAAAAACGGGTGAAGACAAGCATA-3 ′ and primer BB = 5′−) containing a third gene (primer B = 5′-GACAGGGAAAGTGATATAGAAAGGGAGATAGA-3 ′) together with a sudden gene. CAGAGAGAAAAACGGGTGAAGACAAGCATG-3 '), 2mM MgCl₂A 360 bp fragment was amplified from a 50 μL reaction volume of genomic DNA containing. The conditions were 35 cycles of 95 ° C for 3 minutes, 95 ° C for 1 minute, 65.2 ° C for 1 minute, and 72 ° C for 1 minute. The final step was 72 ° C for 10 minutes. PCR products were electrophoresed on a 2% agarose gel using 1 μg / mL ethidium bromide (FIG. 3).
Conclusion
Using the restriction sites recognized by HinfI, the Pit-1 gene (growth hormone factor-1 / pituitary-specific transcription factor required to activate prolactin and GH gene expression) distinguishes two alleles It was done. Two alleles were observed: A, which was not digested, and B, which represents the site. The AA pattern is less frequent than the AB or BB pattern. Milk, protein, and leanness were observed to be significantly superior to the Pit-1AB or AA pattern compared to BB. This indicates that heterozygous animals are more productive and better cows. The percentage of fat is lower in AB than in BB animals and is the result of producing more milk with a nearly constant fat yield.
These results indicate one action of Pit-1. However, at least two effects of Pit-1 were observed using the canonical transformation approach. One is for yield and leanness, and the other is for leanness only. These results can explain that Pit-1 has more than one role through activation of prolactin and GH gene expression. The first role is milk, protein (and fat) yield, and the second role is related to the growth of animal muscles, and the presence of AB increases the leanness and decreases muscularity. I mean.
Interestingly, these findings indicate that canonical transformations are useful in identifying effects on related traits. An association between the Pit-1 polymorphism and the milk traits of dairy cows was shown for the original, but also for the transformed scale. For conformational traits (characters related to milk yield) excluding leanness, the significance of the relationship was low. The canonical transformation also showed that the effect on leanness was only partly the result of a direct effect of Pit-1 on the milk trait.
Furthermore, the identification of specific mutations in the Pit-1 gene allows for a rapid and sensitive method that can be performed to distinguish between various alleles and corresponding traits.

Claims (10)

Use of a genetic marker for identifying a trait for milk production ability or meat production ability from cattle, wherein the genetic marker is located at nucleotide position 1178 of the sequence of SEQ ID NO: 7 in a fragment of the Pit-1 gene . AA, AB or BB allele pattern is observed for the Pit-1 gene with a mutation that is a change from A to G (allele A is an adenine at nucleotide position 1178 of the sequence of SEQ ID NO: 7 And allele B is guanine at the same position) , BB allele pattern is an indicator of the bovine muscle trait, AB allele pattern and AA allele pattern is an indicator of the cow's milk production trait Use of genetic markers. Use according to claim 1 , wherein the observation is carried out via an electrophoresis gel. A method for detecting a trait for milk producing ability or meat producing ability of a bovine associated with a genetic polymorphism of the Pit-1 gene at nucleotide position 1178 of the sequence of SEQ ID NO: 7, comprising :
(1) isolating genomic DNA from cattle;
(2) isolating a fragment from the genomic DNA comprising a fragment of the Pit-1 gene as necessary;
(3) detecting an AA, AB or BB allele pattern of the Pit-1 gene (allele A is an adenine at nucleotide position 1178 of the sequence of SEQ ID NO: 7, and allele B is a guanine at the same position. The BB allele pattern is an indicator of the bovine muscle trait and the AB and AA allele patterns are indicators of the bovine milk production trait) ;
(4) Analyzing the AA, AB or BB allele pattern to determine the bovine trait, and analyzing the AA, AB or BB allele pattern, the bovine muscle and fat traits are milk A method comprising the step of being distinguished from production traits. The method according to claim 3, wherein in step (3), the detection of the allelic pattern in the Pit-1 gene is performed using a restriction endonuclease. 5. The method according to claim 4, wherein the restriction enzyme is HinfI. 5. The method according to claim 3 or 4, wherein the fragment of the Pit-1 gene is isolated using PCR primers. The method of claim 6, wherein the PCR primer is

The way that is. 5. The method according to claim 4, wherein the detecting step further comprises analysis by RFLP or CFLP or SSCP or DGGE after digestion. The method according to claim 3, wherein in step (3), detecting a mutation of a gene polymorphism in the Pit-1 gene comprises a primer covering the mutation in the Pit-1 gene. Method performed using. The method of claim 9, wherein the PCR primer is
For AA genotypes that characterize milk production,

About BB genotype that is characteristic of meat production ability,

(Here, allele A is adenine at nucleotide position 1178 of the sequence of SEQ ID NO: 7 and allele B is guanine at the same position)
The way that is.

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