JP3573130B2 - Highly efficient genome scanning - Google Patents

Description

これらの多型バンドのうち、バルク法で優性ホモと劣性ホモと明確な差が出た遺伝子近傍バンドの候補はRAPDとAFLPではそれぞれ、6本と41本であった。これらのバンドをまず実際のF1株12株を用いて検定し、1次スクリーニングを行った(図5)。さらにF1の株数を増やして行って、最終的にはF1斤株125株での検定でavrPib近傍の精密地図を作製した(図6)。RAPD法では2本、本法では12本のバンドが実際の目的遺伝子avrPibから20CM以内にあることが示された。最も目的遺伝子に近いバンドはRAPD法で得られたものでは5.3CMであったのに対し、本法で得られたものでは1.8CMとはるかに近かった。いもち病菌では1CMは約80kBに相当するので、この距離は物理地図の作製に十分移行できる距離といえる。
［実施例２］イネのカマイラズ変異体遺伝子の一つbc-3の精密マッピング
イネの桿の細胞壁の2次肥厚に必要なセルロース合成ができなくなった変異体(カマイラズ：brittle culm)の一つであるbc-3の原因遺伝子の近傍の核酸マーカーをゲノム走査法で検索した。F2分析のための交配の親株としては、japonica型のbc-3変異体：M11、indica型の野生型Kasalathとの交配を用いた。F3世代までの分析により、変異型ホモ個体10個体、野生型ホモ個体8個体を同定して、それぞれのゲノムDNAを混合したものを、両親株と共にAFLPと組み合わせたゲノム走査法によるバルク分析に供した。それぞれの核酸をEcoR I(6塩基認識），Mse I(4塩基認識)の2種類の酵素で2重消化した後、それぞれの酵素の切断部位に隣接した3塩基の選択ヌクレオチドを持つプライマー1430組み合わせについて、バルク分析によるゲノム走査法を適用した。図7はその1例である。
その結果、97の近傍マーカーの候補が得られ、このうち50が劣性ホモ個体から、マーカーの組み替え体を検出するのに有効に用いることができる。それぞれのマーカー候補について10個体づつの劣性ホモ個体(20染色体)によるF2分析を行ってマーカーの絞り込みを行い、特に24マーカーについて劣性ホモ個体32個体64染色体での分析を行った結果、標的遺伝子と共分離している（遺伝子との距離が0CMの）マーカーが1つ見出された(図7)。
［実施例３］
オオムギ（5.5GB）のようにゲノムサイズが極めて大きな生物では、AFLPにより本発明を適用しようとする場合、ゲノム断片両側でのプライマーの選択塩基数が各々3である場合、イネ（450MB）程度のゲノムサイズの様に非変成条件で2本鎖のままDNAを泳動したのでは必ずしも明確なバンドにならない。この場合、選択プライマーの数を増やすのも一つの対応法であるが、3づつの選択マーカーを用いてもゲルに6〜8.5Mの尿素を加えた変性条件下で、サンプル緩衝液にも50％のホルムアミドを加え、泳動直前に90℃3分の熱変性を加えることによりDNAを1本鎖として泳動すると、十分なバンドの解像度が得られる。
図8はオオムギの2条性を決める遺伝子を探索するため、アズマムギ(6条)×関東中生ゴールド(2条)の掛け合わせにアズマムギ(6条)による戻し交配を7代重ねながら、2条性を示す系統を選んで行き、アズマムギを背景とした2条の準同質遺伝子系統として確立されたものと、戻し親のアズマムギとをゲノム走査法で比較したところである。32レーンで16プライマー対における差を検索したものであるが、両者で差があるものは極めて限られており、これが2条性の遺伝子と強く連鎖した領域にある多型バンドの候補である。
この泳動条件でオオムギでは平均約58本のバンドが同定される。大型のシークエンサーゲルでオオムギのAFLPを行った場合での平均バンド数が約88本（Castilioni et al．1998 Genetics 149：2039-2056)であるので、約67％のバンドが同定されていることになる。大型ゲルでは1日でゲル1枚32レーン程度の効率でしかできないところが、本発明では1日で256レーンの処理ができるので、5〜6倍の効率が得られることとなる。
［実施例４］ゲノム走査法で同定されたイネ品種の識別バンドの単離と配列解析による特異的プライマーの設計
市販の精米の品種を簡便に判別するためのSCARマーカーを開発して、その場合でPCRを行うことにより誰にでも容易に品種を判別できるシステムの構築を試みた。
ゲノム走査法(AFLP)で市販の精米主要10品種を識別するバンドを55種類のプライマーを用いて検索した。この検出にはわずか2日しかかからない。多数の品種判別に適したバンドが得られたが、そのうちの一つで「あきたこまち」に特異的なバンドを幅の広いレーンで泳動後、蛍光色素（vistra green）で染色して切り出し、TEバッファー中で破砕抽出してからAFLP用のプライマーを用いてPCRで増幅した。PCR増幅産物を適当なプラスミドに挿入して大腸菌に導入し、菌を培養増幅して得られたプラスミドに目的のインサートが入っていることを制限酵素で確認後、配列の解析を行い、目的バンドを特異的に増幅するプライマーを設計した(図9A)。このプライマーを用いて、主要10品種から得たDNAをテンプレートとしてそれぞれPCR増幅を行い「あきたこまち」でだけ特異的なバンドが増幅されることが確認された(図9B)。
［実施例５］核酸マーカーに対応するライブラリーのクローンの選択方法
通常、ゲノムライブラリーから本法で得られた特定のバンドに対応するクローンを同定する場合、実施例３のようにして得られたSCARマーカーを利用して、ライブラリーを載せた高密度メンブレンにコロニーハイブリダイゼーションを行ってポジティブクローンを選択することが行われるが、多数のバンドに対して全てこの方式を用いるには手間とコストがかかりすぎ、また必ずしも単離されたバンドの内部配列がユニークであるとは限らない。
マーカーバンドの単離を経ずに直接、ゲノム走査法により特定のバンドに対応するライブラリーのクローンを選択する方法としては、次のようにすればよい。まず、全てのライブラリークローンからBAC等のゲノムクローンのDNAをプラスミド抽出機により抽出して元のクローンのプレートと同じ配列のDNAによるプレートを作製しておく。次に全ゲノムライブラリーを分割して1ゲノム相当以下のサイズのサブライブラリーの集合とする。これにより、1つのサブライブラリーの中で平均1クローンだけが特定のバンドに対応するようになる。さらに、各サブライブラリーを構成する数枚分のマイクロプレートのクローンから行、列、プレートの各番号を座標として同じ座標番号を持つクローンから10ngづつのDNAを集めてゆき、各座標に対応する座標サンプルを作る。例えば、3行目にあるクローンからは、プレート番号や列番号に関わりなくすべて10ngづつDNAを取って集めて、行座標3行目の座標サンプルとする。同様にすべての座標について座標サンプルを作り、各座標サンプルと対照の全ゲノムサンプルについて同じゲノム走査法を適用する。座標サンプルDNAを作製するには、ゲノムライブラリー全クローンのDNAを抽出しなくても、2ml程度に増やした各クローンの培養をそのまま少量づつ行、列、プレートごとに混ぜて、座標サンプルとした後に、その混合培養からDNAを抽出してもよい。対照となる全ゲノムから増幅されたレーンの目的バンドと一致するバンドが行、列、プレートについて見いだせれば、その番号が求めるクローンの3次元座標となり、当該クローンをサブライブラリーから拾い上げることができる。万一、サブライブラリーに多数の該当クローン候補が存在するときは、それらを拾い集めた後に、再度最終決定のためのゲノム走査法を対照と共に行えばよい(図10)。
この手法が力を発揮するのは、ゲノムライブラリーの構成クローンを網羅するように、多数のゲノム走査法で得られるバンドをすべてライブラリークローンに対応させて、全ゲノムのコンティグを作製する場合である。これにより一人でも全ゲノムコンティグの作製が容易に行われる。
図10Aでは、いもち病菌（40MB）のゲノムライブラリー（平均120kB；6ゲノム相当）を1つがほぼ1ゲノムに相当する6個のサブゲノムライブラリーに分割した場合において、6つの半プレート(3枚のフルプレート)からなる1つのサブゲノムライブラリーから特定のクローンを同定する方法を示している。1サブゲノムライブラリーについて、行座標の1行目では6枚の半プレートの1行目のクローン（6列×6半プレート＝36クローン）のすべてからDNAを10μgづつ集めて1行目を代表する座標サンプルとしている。すなわち、1サブゲノムライブラリーでは行、列、プレート軸を代表する座標としてそれぞれ8、6、6の各座標サンプル、計20の座標サンプルでサブゲノムライブラリー全体の8×6×6＝288クローンを同定できる。
図10Bで示すように、この20の座標サンプルをテンプレートとした20レーンと、対照の全ゲノムをテンプレートとした2レーンとを同時にゲノム走査法で泳動することにより、22レーンで容易に対照で得られたバンドに対応するクローンを1つのサブゲノムライブラリーの中から同定することができる。
1枚のゲル(66レーン)では3つのサブゲノムライブラリーを同時に泳動できるので、2枚のゲルで全ゲノム、6サブライブラリーをカバーできる。すなわち、1プライマーペアによる1回のAFLPを考えれば、約25-40本のバンドに対応するクローンの検索が、6ゲノム相当の全ゲノムライブラリーについて2枚のゲルでできることになる。標準のゲノム走査法では4枚のゲルを用いるので、1回の泳動で2プライマーペアにより得られた50-80バンドに対応するクローンが同様に全ゲノムライブラリーから同定できることになる。
これにより控えめに見積もっても1回の電気泳動で2プライマーセットの約50-80バンドをクローンへ対応付けすることが可能であり、25回の電気泳動で約1,200-2,000本のバンドをゲノムに対応付けすることができる。いもち病菌のゲノムサイズが40MBであることを考えると1,500本のバンドが得られたとしてゲノム中でのバンドの平均密度は40MB÷1,500本≒27kB／本となり、120kBの平均的クローンに平均4.4本のバンドが得られることになり、クローンの重複度が平均6あることも考えれば、ゲノムコンティグを作製するには十分過ぎる密度と考えられる。これにより、BACのプラスミドさえ用意できていれば約1ヶ月で6ゲノム相当からなる全ゲノムライブラリーのコンティグが完成する。
プラスミド自動抽出機がフルに使えれば、18プレート分のプラスミドは2週間程度で調製可能である。
産業上の利用の可能性
本発明の方法および電気泳動装置を用いることにより、例えば、下記のような目的のための核酸マーカーを極めて簡便かつ高能率で検索、同定、取得することが可能となった。
▲１▼重要な機能・形質を制御する単一遺伝子をその近傍にある核酸の多型を利用してマークする多型マーカーの開発。
この場合、目的遺伝子を持つ生物に既存のマーカーからなるゲノムマップが存在する必用はない。
▲２▼ポジショナルクローニングにおける標的遺伝子近傍のクローンの同定および単離
重要な機能・形質を制御する単一遺伝子をポジショナルクローニング法により、その染色体上の位置情報だけに基づいて単離・クローニングしようとする場合、その極近傍のマーカーを検索し、BAC(バクテリア人工染色体)等のゲノムライブラリーから遺伝子の近傍領域のクローンをつり上げるためのマーカーを提供する。この場合、目的遺伝子を持つ生物に既存のマーカーからなるマップが存在する必用はない。
▲３▼遺伝分析、高密度地図の作製
ある生物の遺伝子地図を作製するための多数の核酸マーカーを高効率で提供すると共に、それらのF2ないしはRI系統においての連鎖分析を高能率で行い、高密度地図の作製を高い効率で行う。これにより複数の遺伝子の寄与に基づく定量的形質（QTL：Quantitative Trait Loci）の解析も容易に行われる。
▲４▼品種の識別
精米を始めとして、市場に出回っている食品類や種子の品種を識別するためのマーカーバンドを高能率で検索する。さらには、得られた識別バンドを単離・クローニングしてその配列の解析からSCAR(Sequence Characterized Amplified Region)分析用のプライマーを設計する。こうしたSCARマーカーを用いれば、その場で短時間で品種判別を行うことも可能となる。また、特定遺伝子をマークする近傍マーカーもSCAR化することにより、育種等におけるマーカーとして使いやすくなる他、遺伝子近傍のBACクローンを釣り上げるためにも役立つ。
▲５▼生物の全ゲノムをカバーするコンティグの作製
ある生物のゲノムライブラリーを構成するクローンをつなぎ合わせて、全ゲノムをカバーするコンティグを簡便に作製することができる。すなわち、数ゲノム相当からなるゲノムライブラリーを約1ゲノム相当のサブライブラリーに分割した後、各サブライブラリーを構成するマイクロプレートの集合について、その行、列、プレートをそれぞれ、x、y、z軸とする座標を作り、各座標と直交する全てのクローンのDNAをまとめて座標サンプルを作製して鋳型とする。これを通常の全ゲノムDNAを鋳型とする対照とを並べてゲノム走査法で泳動する事により、全ゲノムレーンに見出される各バンドに対応するクローンを同定することができる(図10)。1バンド/20-50kB程度の密度でバンドが見出されれば、平均して数クローンが重複した全ゲノムコンティグが完成できる。 Technical field
The present invention relates to a method for detecting a target nucleic acid by electrophoresing a large number of nucleic acid samples with high efficiency, and an electrophoresis apparatus used in the method. The method of the present invention is particularly useful in the detection of polymorphisms in genomic DNA, genetic analysis, genetic mapping, and the creation of contigs or physical maps covering the entire genome of an organism.
Background art
Until now, when trying to detect differences between biological varieties at the nucleic acid level, techniques such as the RFLP (Restriction Fragment Length Polymorphism) method and the RAPD (Randomly Amplified Polymorphic DNAs) method have been mainly used. However, the RFLP method requires a large amount of a DNA sample and, in addition, in order to search for a marker in the vicinity of a specific gene, a genomic map created based on an existing RFLP marker for the organism is indispensable. In addition, the production of this map requires a long time, a huge cost, and a large number of personnel, and organisms having a genetic map having an RFLP marker at a sufficient density are extremely limited. On the other hand, detection of a polymorphism by the RAPD method is easy for a relatively large number of samples, but the number of bands that can be obtained stably at one time is limited to about several bands. In this case, if the number of bands obtained is increased by relaxing the PCR conditions, there is also a problem that the reproducibility of the obtained polymorphic band is reduced.
Recently, the AFLP (Amplified Fragment Length Polymorphism) method is often used because it can compare many (50-100 or more) bands at once, consumes less DNA of the sample, and has higher reproducibility of the obtained band. It has become. However, this original method requires a lot of experience, such as using a large sequence gel ranging from 40 to 50 cm, detecting nucleic acid bands by autoradiography using an isotope, and requires use conditions. However, there is a disadvantage that detection is time-consuming and that a large number of samples cannot be analyzed (about several tens at a time).
Recently, a technique for relatively easy detection of bands by PCR using fluorescent primers and an automatic sequencer has been developed, but the sequencer used in this technique is expensive (1-2 million yen or more), and However, in the experiment using this method, a machine used for determining the base sequence of a gene is occupied for a long time. Furthermore, since band detection in this method is premised on being performed by a system using an automatic sequencer, a target band cannot be isolated and analyzed after detection. For this reason, a major problem is that the detected band cannot be easily detected with a specific primer as a SCAR (Sequence Characterized Amplified Region) marker. In addition, the fluorescent primers currently supplied are 8 to 9 types, and even when all of them are combined, only the combination of 64-81 primer pairs can be obtained.
The RLGS (Restriction Landmark Genome Scanning) method can also be used as a method to detect a polymorphic band in the entire genome at once.However, this method also uses a radioisotope and requires several days to detect a trace amount of a marker. In addition, it is necessary to handle a huge (40 × 30 cm) gel or a long and thin agarose gel for each sample. For this reason, this method requires strength and sufficient experience. In addition, this method requires the use of a large amount of expensive restriction enzymes in order to perform restriction enzyme cleavage of nucleic acids in an agarose gel, resulting in a problem of high cost.
For example, when the RLGS method is performed on a rice genome (450 MB), only a limited 8-base cleavage enzyme such as Not I can be used for the restriction of the labeling site. The theoretical upper limit of the obtained dot is 450MB ÷ 4⁸= 13,700, and in fact, only 2,000 to 4,000 dots, about 1/3 to 1/6, can be obtained due to the nature of electrophoresis. In addition, since the individuals to be compared are electrophoresed on different gels, the migration patterns often shift, and an expensive large-sized scanner and two-dimensional migration software are required for the mutual comparison.
On the other hand, in the early days of preparing a genomic library in which adjacent clones covering the entire genome were arranged and connected by overlapping each other, attempts were made to use markers of an existing map such as an RFLP map. However, even a so-called high-density map has at most about 2,000 markers, and a small genome (450 MB) as small as rice has an average density of 200 KB / band or more, so it covers the entire genome. Making contigs is too sparse, and searching for this many RFLP markers is enormous, expensive and time consuming.
Recently, fragments obtained by cleaving library-constituting clones with appropriate restriction enzymes are run on a high-resolution gel, all of the patterns are digitized and imported as a database. A method of assembling a contig covering the genome is often used. However, this technique also requires enormous labor and cost to perform autoradiography by radiolabeling the end after cutting all clones.
Disclosure of the invention
The present invention has been made in view of such circumstances, and a purpose thereof is to efficiently and inexpensively electrophorese a large amount of a nucleic acid sample, to detect a target nucleic acid, and to provide a method for the method. An electrophoresis apparatus is provided. In a preferred embodiment of the method, the present invention provides a method for detecting a genomic DNA polymorphism using the method, a method for genetic analysis, a method for preparing a genetic map, a method for identifying a genomic clone for a specific band, and a method for whole genome. Methods for making an ordered collection of contigs that cover
The present inventors have conducted intensive studies to solve the above problems, and as a result, an electrophoresis method using a small gel, which has been generally used for protein electrophoresis, has been able to efficiently remove a large amount of nucleic acids. It was thought that electrophoresis could be used to detect the target nucleic acid.
Thus, the present inventors have developed an electrophoresis apparatus for electrophoresing nucleic acids, which can be equipped with a plurality of 10 to 30 cm square gel plates at the same time to perform electrophoresis, and A unique electrophoresis device capable of simultaneously electrophoresing 32 or more nucleic acid samples was used. Using this electrophoresis device, a nucleic acid marker near the non-pathogenic gene of rice blast fungus and the rice camillaz mutant gene Was searched. As a result, it has been found that the polymorphic band can be detected by this method with remarkably high efficiency as compared with the case where the search is performed by the conventional RAPD method.
In addition, as a result of performing a linkage analysis on the detected polymorphic bands, among the detected polymorphic bands, in particular, a band positioned near the target gene, according to this method, far more than the conventional method. Was found to be efficiently obtained.
Furthermore, this method was shown to be useful in isolating and specifically amplifying various important polymorphic bands found by the above-mentioned method. As an example, the present inventors have isolated a large number of bands identifying 10 varieties for rice milling by this method, actually designed primers based on the sequence information of bands specific to Akitakomachi, PCR was performed using genomic DNA obtained from 10 cultivars as templates. As a result, they found that a specific amplification product by PCR was detected only in Akitakomachi. That is, it was shown that according to the method of the present invention, a polymorphic band for easily distinguishing rice varieties can be efficiently obtained.
Furthermore, the present inventors have found that, according to this method, it is also possible to prepare a genetic map of an organism or to obtain a nucleic acid marker for a contig covering the entire genome of an organism.
Accordingly, the present invention relates to a method for efficiently and inexpensively electrophoresing and detecting a large amount of a nucleic acid sample, an electrophoresis apparatus for the method, and use thereof, and more specifically,
(1) A method for electrophoresing nucleic acids,
(A) Using an electrophoresis apparatus capable of simultaneously carrying out electrophoresis by equipping a plurality of gel plates of 10 to 30 cm square and simultaneously carrying out electrophoresis of 32 or more nucleic acid samples per gel plate. Electrophoresing the nucleic acid sample, and
(B) detecting a nucleic acid band on the gel after electrophoresis,
(2) The method according to (1), wherein the electrophoresis is performed using a discontinuous buffer gel.
(3) The method according to (1), wherein the nucleic acid sample is single-stranded DNA prepared by dissociation of double-stranded DNA by denaturation treatment, and electrophoresis is performed using a denaturing gel.
(4) The method according to (1), wherein the band of the nucleic acid on the gel is detected by fluorescent staining or silver staining.
(5) The method according to any one of (1), (2), and (4), which is performed to detect a genomic DNA polymorphism between test individuals.
(6) The method according to (3), which is carried out to detect a genomic DNA polymorphism between test subjects.
(7) The method according to (5), wherein the nucleic acid sample is a DNA fragment amplified by the AFLP method.
(8) The method according to (5), wherein the nucleic acid sample is hetero double-stranded DNA.
(9) A method for preparing a DNA fragment exhibiting a polymorphism, comprising a step of isolating a DNA fragment exhibiting the polymorphism detected in the method according to any one of (5) to (8) from a gel. Method,
(10) a DNA fragment showing polymorphism between test subjects isolated by the method according to (9),
(11) The method according to any one of (1) to (8), which is performed for performing a genetic analysis.
(12) The method according to (11), wherein the genetic analysis is F2 analysis, RI (recombinant imbred) analysis, or QTL (Guantitative Traits Loci) analysis.
(13) The method according to any one of (1) to (8), which is performed for creating a genetic map of an organism,
(14) a genetic map of an organism created using a genomic DNA band showing a polymorphism detected by the method according to (13) as a marker,
(15) A method for selecting a clone corresponding to a specific nucleic acid band on a gel detected by the method according to any one of (1) to (8) from a genomic DNA library,
(A) dividing a genomic DNA library of a specific organism into a plurality of sub-libraries each having a size of one genome or less of the organism;
(B) A step of assigning the row, column, and plate numbers of the sublibrary to all clones included in each sublibrary (here, the row, column, and plate are coordinate X, coordinate Y, and coordinate Z, respectively) ,
(C) Clones showing a particular row of all plates (coordinate X clones) and clones showing a particular column of all plates (coordinate Y clones), and on a particular plate of one sublibrary 5. A method according to claim 1, wherein all clones (coordinate Z clones) are collected and DNA is separately extracted to prepare a coordinate sample, a genomic DNA prepared from the organism is prepared as a control, and the coordinate sample and the control are arranged side by side. Electrophoresis by the method described in any of the above, a step of detecting a band,
(D) The band at the same coordinate among the clones of the coordinate X clone group, the coordinate Y clone group, and the coordinate Z clone group corresponds to the band of the nucleic acid of interest in the control and the band having the same electrophoretic mobility on the gel. Determining whether or not
(E) selecting a clone corresponding to the determined three-dimensional coordinates from the sub-library.
(16) The method according to (15), which is performed to create a contig covering the whole genomic DNA of a specific organism.
(17) An electrophoresis apparatus for electrophoresing nucleic acids, wherein a plurality of gel plates having a size of 10 to 30 cm square can be simultaneously mounted to perform electrophoresis, and more than 32 nucleic acids per gel plate An electrophoresis apparatus capable of simultaneously electrophoresing a sample.
1. Electrophoresis and electrophoresis equipment
The electrophoresis apparatus of the present invention is an electrophoresis apparatus capable of mounting a small polyacrylamide gel (10 to 30 cm square, standard 18 cm square). It is possible to electrophores 2 (4-4 standard) size markers at the same time, and by electrophoresing 2 or more (4 standard), multiple (265 standard) samples can be electrophoresed simultaneously. It is possible to do. One example of the electrophoresis apparatus used in the present invention is shown in FIGS. 1 to 4.In this electrophoresis apparatus, 64 samples and two sizes are formed by 66 wells of a 1 mm thick gel made of one piece of 18 cm square glass. The standard and the standard can be run simultaneously, and four gels can be electrophoresed simultaneously with one electrophoresis device. Thereby, 256 samples can be simultaneously electrophoresed at one time.
For the gel, a discontinuous polyacrylamide electrophoresis system (Laemmli, UK (1970) Nature 227: 680-685), which is usually used for protein electrophoresis, can be used to reduce a sample of up to 10 μl to a narrow width of 1 mm. Can be electrophoresed with high resolution even in the lane, and the sensitivity of band detection is also improved.
In the nucleic acid electrophoresis method of the present invention, in order to improve the resolution of the nucleic acid band on the gel, concentrated gel (Tris-HCl pH6.8, 0.5M) and separation gel (Tris-HCl pH8.8, 1.5M) to perform two-layer electrophoresis. Regarding the electrophoresis of the nucleic acid, it is possible to migrate the nucleic acid as a double strand, or to electrophores the nucleic acid as a single strand after denaturation according to the purpose. In this case, electrophoresis is performed on a denaturing gel (a gel containing 6 to 8.5 M urea). When a nucleic acid polymorphism is detected, a small polymorphism that cannot be normally detected can be detected with high sensitivity by electrophoresis as a heteroduplex.
In the nucleic acid electrophoresis method of the present invention, it is preferable to use a silver staining method or a fluorescent staining method for detecting a nucleic acid band after electrophoresis. If silver staining is used, highly sensitive detection can be performed in a short time (1-2 hours). Moreover, compared to the method using an isotope, less experience is required, and if it is safe and dried as it is, it can be stored as a sample and sensitivity can be further increased. If fluorescent staining is used, the results can be seen in about 30 minutes of staining, and the nucleic acid recovery rate is high and excellent for excision and recovery of bands.
In the method of the present invention, for example, an amplified DNA sample or the like is designed by designing an electrophoresis gel comb so that two or more lanes can flow at the same interval (9 mm) as the holes of an 8 × 12 96-well microplate. Can greatly improve the operation efficiency when loading the gel on the gel.
2. Detection of nucleic acid markers
In the case where a polymorphism of a nucleic acid is to be detected by the method of the present invention, the most efficient method is used in combination with AFLP (Amplified Fragment Length Polymorphism: Vos P, et al. (1995) Nucleic Acids Research 23: 4407-4414). Although considered to be high, it can be combined with various other nucleic acid polymorphism detection methods.
For example, if the AFLP method is used, in the case of a rice (450 MB) -sized genome, primers for amplifying a genomic fragment having both a 6-base cleavage side and a 4-base cleavage side on both sides are selected by 3 bases on both sides. When nucleotides are used, about 50 amplified bands are obtained per lane (see formula below). In a standard system consisting of 4 gels and 256 sample lanes, about 12,800 bands are obtained in one run (Example 2).
Whole genome size ÷ number of cuts by 6 base restriction enzyme 選 択 selectivity by 3 nucleotides on both sides of genome fragment = number of bands
(450MB ÷ 4⁶× 2 ÷ 4^3x2≒ 50)
This is comparable to the information content of several gels when performing the RLGS method, and since the lanes to be compared can be arranged next to each other, it is easy to directly use raw data without using a special reading device etc. Can be compared to
As shown in FIG. 6 (Example 2), from the experimental example of actual AFLP obtained by cutting rice genomic DNA with EcoR I and Mse I, the number is close to the number estimated by the above calculation. Although the number of bands is obtained, the number of particularly clear bands is about half.
Performing PCR with 5-10 μl using a 0.2 ml microplate for 96 samples can reduce the cost of enzymes such as heat-resistant DNA polymerase and nucleotides, and use 8 10 μl pipettes at a time. Since the sample can be transferred to a gel, the efficiency is high, and electrophoresis of four gels can be performed once a day with sufficient margin.
The four gels after electrophoresis can be stained efficiently and at low cost by performing silver staining in one container at a time. Generally, a commercially available silver staining kit for proteins can be used for staining.
As a nucleotide primer used in the AFLP method, a nucleotide compatible with any restriction enzyme can be used, and an arbitrary selected sequence of one to several bases can be added to the 3 ′ side, so that it is almost infinite. Becomes possible. For plants with a genome size of 1 GB or less, using EcoR I and Mse I as the respective restriction enzymes of 6 bases and 4 bases, and using PCR primers each having 64 3 nucleotide selection sequences, 64 × 64 = 4,096 amplifications are possible, and in genetic analysis between parents with a polymorphism rate of 5 to 10%, 10,000 to 20,000 polymorphic bands can be searched. This is a practically sufficient number for creating a physical map, and exceeds the number of markers in a conventional genetic map by one or more digits.
Important bands such as bands near the target gene obtained by bulk analysis (Michelmore RW, et al. (1991) Proc. Natl. Acad. Sci. USA 88: 9828-9832) and the like were cut out after staining, and gels were cut out. After crushing, it is extracted with an appropriate buffer, and then subjected to PCR again, inserted into an appropriate plasmid, ligated, and isolated to analyze its sequence (Example 4).
With the above combination, for example, four lanes / primer pair electrophoresis were carried out, for example, two samples obtained by mixing the parents of the cross and two dominant and homozygous homozygous individuals in F2, each consisting of about 10 individuals. For example, a standard electrophoresis apparatus (4 gels: 256 lanes) can run 64 primer pairs in one run. As a result, all combinations of the 4,096 selection primers are completed in 64 runs (about 2 months in total). This is equivalent to scanning and retrieving 20,000 polymorphic bands in the whole genome in a combination having about 10% polymorphism such as the cross between rice and India. This efficiency is one to two orders of magnitude higher than conventional techniques, which allows for the isolation of genes in a short time, with or without a genetic map. In addition, the cost for performing all of the 4096 searches is inexpensive at around 400,000 yen.
3. Genetic analysis
1) F2 analysis
When trying to determine the genetic distance (or distance on the map) between any gene or combination of polymorphic markers, the most commonly used is F2 analysis, but the method of the present invention is used. According to the F2 analysis described above, the distance between polymorphic markers or between a polymorphic marker and a gene can be determined extremely efficiently.
In other words, similar to general F2 analysis, a line (A) that has a certain gene and a line that does not have the target gene or has a clear difference in its traits. Select and cross with a certain line (B) to obtain a large number of F2. The number of F2 individuals to be analyzed depends on the accuracy of the analysis.In order to obtain the accuracy of 1CM, if 50 homologous individuals (usually recessive homologous individuals) are used, the target gene and polymorphic marker of 100 chromosomes are used. The genetic distance between the two can be determined by examining the recombination rate between them. Analysis of recessive homozygotes shows that if there is no recombination, all individuals with the same marker type as the parent with the recessive trait in one parent have a recombination rate of 1/100, and only one chromosome of one individual shows recombination. Will be done.
In the present invention, since the standard system can analyze 256 individuals at a time, if only such homologous individuals are obtained, it is possible to test the recombination of the polymorphic marker on 512 chromosomes by one run, Analysis with an accuracy of 0.2CM is possible. By using the AFLP method as a polymorphic marker, the test is performed with a very small amount of sample DNA (100 ng or less). That is, genomic DNA is prepared from each individual, double digested with two enzymes, EcoR I and Mse I, and an adapter suitable for each enzyme is bound to the genomic DNA fragment. Primary PCR is performed using primary primers compatible with this adapter to amplify genomic fragments. Next, using a secondary primer having an arbitrary 1-4 bases extended to the 3 ′ side of the primary primer, only a part of the genomic fragment adapted to this sequence was subjected to PCR amplification, and the molecular weight was determined by the electrophoresis method of the present invention. Separate according to. After the electrophoresis, the gel is stained by silver staining or the like, and a test is performed to determine whether or not the target polymorphic marker has recombination.
According to the present invention, precise F2 analysis at a level of 0.2 CM for 256 individuals was completed with a small amount of DNA in one run, and no blotting or autoradiography was required, and four stained and dried gels were used. Can be stored in the album as it is in cabinet size, so the analysis of the results is extremely easy.
2) RI analysis
RI strains (Recombinant Inbred lines) refer to strains obtained by self-breeding F2 individuals produced by crossing different strains for generations. As a result of the large number of inbreds, most loci are homozygous, and the number of heterozygotes is small, so the segregation ratio is 1: 1 for the dominant trait: recessive trait. Therefore, the genetic analysis according to the present invention can be performed as it is. The ratio of heterozygous individuals is halved, and a more precise genetic analysis is possible than F2 analysis, in which the separation ratio between the dominant and recessive traits is 3: 1. If an appropriate RI strain is available, genetic analysis (RI analysis) using genomic DNA extracted from many RI strains using the present invention can be performed in exactly the same way as in the case of F2 analysis.
3) Narrowing down nearby markers
By using the electrophoresis method of the present invention, in order to narrow down the markers near the target gene obtained by the bulk analysis method by F2 analysis and finally map the nearest marker group, once by the standard method, Since 256 samples can be electrophoresed, genetic analysis can be performed with extremely high efficiency. In other words, in rice (in the case of the Japanese-Indian cross), as described in Section 2, if the polymorphic markers of 20,000 bands are evenly distributed in the whole genome of 2,000 CM, the gene obtained by the bulk method It is expected that the number of markers present in a region of a total of 20 CM, about 10 CM each on both sides in the vicinity, is 200 bands. In order to narrow down the nearest markers among these nearby markers, as a first step, first, 14 recessive homozygous individuals of the F2 generation per marker and the DNA of their parents were flown to 16 lanes per marker, The presence or absence of recombination between the gene and the candidate marker is examined. In this way, the recombination rate for 28 chromosomes is calculated for each marker. The resolution at this time is about 3.5 CM. As a result, four bands of candidate markers are tested per gel, and it is possible to check 16 bands of markers in one run, and at the same time, individuals that have undergone recombination in the vicinity of the gene are clearly identified. It becomes. After that, only eight lanes are used in total, including six transgenic individuals and parents in the vicinity, and the check of the remaining 192 markers is completed by six runs.
Assuming that the marker and the position of chromosome recombination occur evenly, it is expected that the above-mentioned screening will provide about 40 bands of markers near 8CM in total, within about 4CM on both sides of the gene. In the second step, in order to map each marker remaining in the primary screening with an accuracy of 1 CM, first of all, 12 markers were used for each run of 3 rounds using a gel, and 62 recessive homologous F2 generations were used. + Run AFLP product for parents. As a result, a precise map of the region within the gene vicinity 8CM is obtained, and individuals that have undergone recombination in the nearest neighborhood within about 1CM of the gene are revealed. Analyzing the remaining 28 bands using these recombined individuals in the vicinity will reveal their locations on this map. Again, the analysis in the vicinity of the gene is completed by checking the 224 lanes with 8 lanes x 28 combinations = 224 lanes together with the 6 nearest recombinants and the parents in combination.
In other words, the nearest marker within about 1 CM can be narrowed down by about 11 times of migration from 200 neighboring marker candidates selected by the bulk method from 20,000 polymorphic bands.
In gene isolation, if an organism with a genome size of 1 GB or less has such a marker, the average 1CM will be 500 kB or less. Can be selected to form a contig.
Even when the accuracy is further improved and analysis is performed at the 0.2 CM level using 256 recessive homozygotes, if the appropriate neighboring marker 4 bands are selected and run four times, which individual It becomes clear whether recombination is occurring nearby. Using the recombined individuals in the vicinity, if the state of recombination in the band considered to be the nearest is checked in the same way as at the second stage, the nearest marker can be easily obtained by one run .
The principle here is based on the assumption that the bands obtained by electrophoresis are distributed almost uniformly in the genome and chromosome, and that chromosome recombination occurs uniformly on the genome. Although the distribution of bands and recombination positions in the chromosome is actually unevenly distributed, the distance between the target marker and the neighboring marker obtained for the number of F2 used is more uniform than in the case of a uniform distribution. Tends to be large.
4) Application to marker breeding
In addition, the near marker of the specific trait gene obtained as described above is used as a highly reliable marker even when performing native breeding by mating, and the marker is used in a large number of mating progeny obtained by mating. Even when performing analysis, analysis by the genome scanning method can easily analyze a large number of individuals by taking only a small amount of DNA sample, and the work efficiency is significantly higher than the conventional method by the RFLP method. Improvement and cost reduction can be achieved.
5) Application to QTL (Quantitative Traits Loci) analysis
QTL refers to a genetic trait locus whose trait expression is not as qualitatively strong and in many cases multiple loci are involved in the expression of that trait. QTL analysis analyzes which locus at which position on the chromosome contributes to the expression of that trait.
In the QTL analysis, polymorphisms in a large number of mating progeny individuals must be examined for a large number of nucleic acid markers uniformly distributed throughout the entire genome. For example, if QTL analysis is performed using markers distributed in the whole genome of 2,000 CM at a marker density of about 10 CM / line, it is necessary to examine 200 markers for F2 of at least about 50 individuals. If this is performed using RFLP markers, it is necessary to perform 200 hybridizations on the membrane on which the nucleic acids of 50 individuals are plotted, and one hybridization takes two days. Even if it does, it will take a total of 100 days. Even if a membrane can be used ten times, the labor required to collect as many as 100 μg of nucleic acid from all 50 individuals to prepare 20 membranes is enormous.
By using the AFLP method in the method of the present invention, several polymorphic bands can be obtained per lane in a sample showing about 10% polymorphism such as the cross between rice and Japanese. The search for 200 markers can be completed by electrophoresis with a little over 50 primer pairs. Since it is possible to run 5 primer pairs in one run (50 x 5 = 250 lanes), migration of 50 individuals can be completed for all markers after only 10 runs (10 days in total). You can do it. Moreover, the amount of 1 μg of DNA required for each individual is sufficient.
Specifically, first, it is necessary that a genomic map of the organism by the AFLP marker has been prepared. Can be easily created by using the present invention as described in (1). From this map, a marker is selected at a desired density, but it is efficient to select as few primer pairs as possible so as to cover the entire genome.
Regarding the crossover, an appropriate polymorphism frequency can be obtained by selecting combinations that are genetically separated from each other in a line that has a remarkable quantitative trait and a line that hardly shows the trait. If the combinations are too far apart, the markers may not be separated smoothly. Approximately 50 individuals (the number of which varies according to the purpose) obtained from this crossing are used to quantitatively measure the target traits for the F2 or RI strains, and use genomic DNA prepared from each for 3.1). As described above, the genomic fragment is amplified by the AFLP method, and is electrophoresed by the system of the present invention to check and record the marker band polymorphism.
By plotting the product of the number of target traits and the number of polymorphisms of one parent for each band for all marker bands on the map, the contribution of the loci near each marker to the target trait becomes apparent.
4. Application to variety / strain discrimination
According to the present invention, it is possible to efficiently search for a marker necessary for discriminating a specific breed or line of an agricultural or livestock product or various biological species. That is, AFLP is performed using DNAs obtained from a large number of varieties to be compared, and by performing electrophoresis simultaneously using the electrophoresis apparatus of the present invention, tens of bands are obtained for tens to hundreds or more varieties. Can be compared at the same time. This makes it possible to easily select a specific identification band for many types.
In this case, by combining n bands, it is theoretically possible to identify up to 2n varieties / lines, but in the actual case, the number of identifiable varieties / lines is less than this Somewhat less.
When varieties and strains are very close to each other and polymorphism is difficult to obtain with normal AFLP etc., AFLP and RAPD products etc. based on the genome to be compared are mixed, heated and cooled, and migrated as heteroduplex, By comparison with the target band, differences between varieties can be detected with high sensitivity, easily, and efficiently.
If a specific band with high discriminating ability is identified, the band is isolated by the method described in section 7 so that PCR amplification can be performed from specific primers.After PCR, simple agarose electrophoresis is performed for 1 hour. The type can be determined by the degree. Furthermore, a specific fluorescent label is pre-applied to the primer, and whether or not a band is amplified therefrom is checked with a PCR device equipped with a fluorescence detector. It is also possible to identify varieties and strains without any need.
5. Genetic mapping of organisms
When trying to create a genetic map (more precisely, a nucleic acid marker map) that covers the entire genome of an organism, depending on the required map resolution, several hundred to over a thousand markers, as with QTL analysis It is necessary to analyze using a number of F2 individuals. In the case of higher plants, especially the major crops, most of which have a total genome length of 1,500 to 2,000 CM, if it is attempted to create a genomic map with the density and accuracy of about 1 CM by the conventional RFLP method, more than 100 F2 generations It is necessary to repeatedly blot the prepared membrane for about 1,800 markers, and it takes four days including preparation of the probe for one blotting.Therefore, even if four kinds of probes are processed at one time, a total of 1,800 Takes days work. In addition, the amount of nucleic acid required for this is enormous, and each individual F2 requires 1 mg or more of DNA. For this reason, genetic maps have been created by teams of tens of people over the years.
In the present invention, if a combination of mating parents having about 10% polymorphism is taken as an example, an average of 4 to 5 polymorphisms can be seen in one primer combination, so that in a preliminary test, 6 or more polymorphisms are obtained in advance. By selecting a primer combination that indicates, and using this to perform mapping using the F2 generation of 128 solids with the standard model, it is possible to map 12 or more markers with 2 primer pairs in one run . Therefore, a map with 1,800 markers can be created with a single person and a total of 150 days of analysis to produce a precise map, which is more than 10 times as efficient as the conventional method. If it is a map of the size of a marker, it can be made in about one month alone.
As a specific procedure, two lines separated by an appropriate genetic distance are crossed to produce a number of F2 individuals or RI lines according to the accuracy of the target map. If the average polymorphism rate does not exceed 10% between both lines, there is little risk of hybrid sterility or distortion of the separation ratio. For the time being aimed at an accuracy of about 0.5 or 1 CM, sufficient accuracy can be obtained with 128 or 64 individuals. Genomic DNA is prepared from these individuals, and the PCR fragment amplified by the AFLP method is electrophoresed, stained, and analyzed by the system of the present invention in the same manner as in the F2 analysis and the RI analysis described above. That is, the prepared genomic DNA is stored in a 96-well microplate, a part (about 0.1 ug) is taken, double digested with EcoR I and Mse I, and the adapter is joined to the cut surface. Perform pre-amplification with the same 5'-side primer as in. Next, the pre-amplified PCR product is used as a template for secondary amplification with a selection primer having an appropriate number of selection bases, and then applied to the electrophoresis apparatus of the present invention. At this time, if 8 or 12 pipettes or 8 to 12 micro syringes are used, the efficiency of gel loading is improved. For 64 samples, 4 primer sets can be used to run about 20 markers, and for 128 samples, 2 primer sets can run 10 markers at a time. For analysis of polymorphic bands, it is efficient to use a gene map creating software such as MAPL or MAMMAKER.
As a reference example, using the system of the predecessor of the present invention using 8 gels of 15 lanes, an ALP of barley barley x RI of Kanto Nakasei Gold, an AFLP map of 227 markers was created in about 2 months using 99 lines, An example is shown in which a map composed of 272 markers was created by integrating the map composed of 45 markers obtained up to now (FIG. 11). Even in this case, an AFLP map can be prepared alone in about two months, and an efficiency about 40 times as high as that in the case of preparing a map using an STS marker or the like has been achieved. In the method of the present invention, the working efficiency is further improved over the system of the predecessor.
6. Application to species with large genome size
The present invention seems to be most effective when used in combination with AFLP, but the characteristics of AFLP are genomic fragments double digested with EcoR I (6 base recognition) and Mse I (4 base recognition) In the first amplification, this adapter sequence was used as a PCR primer so that all fragments were amplified.In the next secondary amplification, the primary primer was used as the primary primer. Using a selection primer having an extension of a selection sequence of one to several bases on the 3 'side of the primer, only a genomic fragment having a sequence compatible with the selection primer at both ends is specifically amplified. In the case of an organism having a relatively small genome size (such as rice: 450 MB), a primer to which a selection sequence of three bases is added on each side of the EcoR I and Mse I sites is used as a secondary primer. As a result, about 50 bands on average are selectively amplified as described in “2. Detection of nucleic acid marker”. For genomes of the size of filamentous fungi such as blast fungus (40 MB), 50 or more (40 MB / 450 MB) x 16-70 bands can be obtained by adding only one base at any restriction site. Is expected to be obtained. On the other hand, in an organism having a large genome size, it can be basically dealt with by increasing the number of bases of the selected sequence in the AFLP primer from 3 to 4.
However, as another method, the conditions for electrophoresis can be changed. Usually, when electrophoresing PCR amplification products, it is convenient to electrophores these materials as a double strand under non-denaturing conditions, but for organisms with a larger genome size (eg, barley: 5.5 GB) If the electrophoresis is performed with this strand, the bands will not be clearly separated. Here, using a denaturing gel containing 8.5 M urea, the DNA sample was subjected to heat denaturation at 90 ° C. for 3 minutes in the presence of 50% formamide to dissociate it into single-stranded DNA. Many bands can be clearly identified (Example 4).
In actual situations, AFLP becomes more difficult as the genome size increases, so it is realistic to use a combination of the above two methods.
7. Isolation of important band and its conversion to SCAR marker by sequence analysis
In the method of the present invention described in 3., 4., 5., etc., a band that carries particularly important information is identified in each case. That is, a band near the specific gene in 3., a band suitable for cultivar discrimination in 4., a band that becomes a landmark in the genetic map in 5., and the like.
These bands are cut out of the gel, eluted by crushing extraction, freeze-thawing, electrophoresis, etc., and then PCR-amplified again with the same primer pair, inserted into an appropriate cloning vector, sequenced, and both ends are primer-primed. (Sequence Characterized Amplified Region) marker (Example 3). In this case, it is easier to extract nucleic acids by using fluorescent staining such as cyber green for staining. Compared to the AFLP method using a sequencer, although the band can be identified but cannot be extracted, the method of the present invention is simple but can fully utilize the important functions of the original AFLP. Are better.
8. Isolation of genomic clones containing specific bands
Bands carrying important information indicated in 3., 4., 5. etc. may require isolation of genomic clones containing them. In particular, when isolating a gene having an important function by positional cloning, identify the nearest markers on both sides of the gene using the methods described in 2. and 3., and then use them to construct a BAC library. A clone carrying a band is selected from a genomic library such as, and the subsequent continuous clone is identified using markers at both ends of the clone (walking), and markers on both sides of the gene (flanking marker It is necessary to create a contig of a series of clones that connect between ().
If a high-density membrane has been prepared in which the constituent clones of the genomic library have been sorted and joined by colony hybridization, bands that have been directly PCR-amplified as in step 7, or bands that have been amplified as SCAR markers The target clone can be picked up by performing hybridization on the membrane using as a probe.
Alternatively, after the genomic library of the organism is divided into subgenomic libraries by the method described in Section 9, clones in the subgenomic library are cloned in such a manner as to correspond to the row, column, and plate number of the microplate. After preparing mixed coordinate markers, performing AFLP on these with the same primer pair using genomic DNA as a control, and then running with this system, the same band as the control is amplified. From the row and plate coordinates, it is possible to identify a clone having the target band without relying on hybridization.
9. Creating a contig covering the entire genome of an organism
If it is possible to clarify the adjacent state of all the constituent clones based on the genomic library of a certain organism by BAC, etc., connect the individual contigs to develop a contig (physical map) covering the entire genome Can be. The physical map created in this way corresponds to a so-called reproduction of the structure of the genome of the organism, and the completion makes it extremely easy to isolate and identify important functional genes without waiting for the entire genome to be sequenced. It will be able to handle the genome of the organism as if it were picked up. In fact, once the whole genome contig is completed, the subsequent genome sequencing can be almost completely automated. However, until now, the creation of whole genome contigs could only be handled by a large research group with dozens of people. However, according to the present invention, it is possible to produce a contig covering the whole genome in about one year by one person with a genome size of about rice (about 500 MB).
Until now, the biggest reason that whole-genome contigs were difficult was that it was difficult to obtain specific labels for marking each clone constituting the genomic library. In the present invention, by combining with AFLP, it is possible to obtain as many as about 30 to 50 highly specific bands at one time in one lane, which are labeled with two parameters of the three base sequences at both ends and the number of bases (length). . All of these specific bands can correspond to the constituent clones of the genomic library, and the distribution density of the bands can be made sufficiently smaller than the length of the BAC clone, thus connecting clones sharing the same marker. It is not difficult to go on.
Here, in order to make a one-to-one correspondence between the AFLP band and the library clone, a genomic library of the whole genome, which is usually several genomes or more, is divided into several sublibraries corresponding to about one genome. Furthermore, the clones that make up the sublibrary are uniquely determined by the row plate, plate, and plate numbers and coordinates, so clones that have the same axis coordinates using the row, column, and plate numbers as coordinate axes respectively A small amount of DNA is collected and mixed from to produce a coordinate sample representative of the row, column, and plate axis coordinates. Using these as a template and performing the genome scanning method of the present invention, comparing the AFLP pattern at both ends of the coordinate lane with the whole genomic DNA as a template as a control, clones corresponding to a specific band using genomic DNA as a template Can be easily detected from the sublibrary. If the subgroup has 2 or n corresponding clones, 2^Three8 or n^ThreeThe clones correspond to the band. In this case, if these eight clones are taken out and subjected to a second electrophoresis by a genome scanning method, two true clones can be identified. A specific method for preparing a coordinate sample of the sublibrary is shown in Example 5.
In this way, if the library has a genome size of about the blast fungus (about 40 MB) and an average insert size of 120 kB, the sublibrary equivalent to about 1 genome will be about 300 clones, 8 rows, 6 columns, 6 half plates Can be stored in In this case, even if a total of 22 lanes of the control genome and 2 lanes of the control genome are flown with respect to 20 lanes of the coordinate sample, the sub-library corresponding to 6 genomes, that is, the whole genome library can be migrated using the two gels. That is, if two primer pairs and about 60 bands can be identified and corresponded in one run, 1500 bands can be handled in 25 runs. This is a density of 40 MB / 1500 lines = 27 kB / line and corresponds to an average of 4.4 bands identified for a BAC clone having an average size of 120 kB. Given this, it can be seen that it is fully expected that almost the entire genome will be covered by this (see Example 5).
For a library equivalent to 6 genomes with an average insert size of 150 kB even for a rice genome size (450 MB), a matrix of 16 rows, 12 columns, and 16 x 2 plates using 32 plates equivalent to almost 1 genome , It is possible to perform electrophoresis for six sublibraries at one time, and about 25 bands are identified at one time. In 200 runs, 5000 bands were identified, which corresponds to a density of 450 MB / 5000 = 90 kB / line, so a 150 kB average clone from a library containing 6 times the degree of duplication had a long contig. It is considered that the density is sufficient to produce.
[Brief description of the drawings]
FIG. 1 is a front view and a top view of a main body of a standard electrophoresis apparatus used for a genome scanning method. Four 18 cm square gels can be run simultaneously. One gel has a comb for loading 66-68 specimens of 1 mm width, and a well for sample application is prepared. Since 2 to 4 lanes are used for flowing molecular weight markers, etc., 64 samples / gel, 4 gels Can run 256 samples at a time. By using eight micropipettes, a sample efficiently amplified by PCR can be applied to a gel.
FIG. 2 is a view showing a gel plate stopper of a standard electrophoresis apparatus used for the genome scanning method.
FIG. 3 is a diagram showing a comb of a standard electrophoresis apparatus used for the genome scanning method.
FIG. 4 is a photograph showing a finished product photograph of a standard electrophoresis apparatus used for the genome scanning method.
FIG. 5 is an electrophoretogram showing primary screening using a genome scanning method for candidate markers near the non-pathogenic gene avrPib of rice blast fungus obtained by bulk analysis. Genome scan combined with AFLP for the parent strains avrPib-, +, bulk avrPib-, +, F1 strain avrPib-6, and +6 for primer pairs A, B, C, and D having four types of nearby marker candidates, respectively. Analysis was carried out. Triangles in the figure indicate bands of marker candidates. For A, B, and C, the primary screening all showed genotype and cosegregation as expected, but for D, recombination was observed for each of the + strains (solid triangles). Note that 5-60 bands were obtained for each primer pair. One gel can scan 3-4,000 bands, and four gels can be run at a time, so that 12-16,000 bands can be scanned. The number of selected nucleotides of the primer pair used here is 3 bases on the EcoRI side and 1 base on the MspI side. The polymorphism rate between the hybrid strains used here was about 5%, and the total genome of the blast fungus was about 500CM, so that about 500 polymorphic bands were obtained with 256 primer pairs, and 1CM / The whole genome can be covered by the density.
Fig. 6 is a detailed map of the vicinity of the gene obtained by mapping the marker near the non-pathogenic gene avrPib of rice blast fungus obtained by RAPD method and genome scanning method using 125 F1 strains by each method. Is shown. As shown in Table 1, the RAPD method used 700 primers, and the genome scanning method searched for nearby markers by PCR of 251 primer pairs. The search and mapping of RAPD markers took three months, while the genome scan method was completed in January. The number of bands counted is limited to very clear ones, so it is quite a few. An: Marker obtained by the genome scanning method. (Rn): Marker obtained by RAPD method.
FIG. 7 is an electrophoresis photograph showing an example of bulk analysis for searching for a marker adjacent to the rice cellulosic mutant Kamylaz: bc-3. From the left, a set of four lanes includes a mutant parent strain (M11: japonica), a mutant (recessive) homo bulk, a wild-type (dominant) homo bulk, and a wild-type parent strain (Kasalath: indica). Arrows indicate candidate markers near the gene where a clear difference is observed between the bulks. Even in the case of the clear band alone, 20-30 lines are counted per lane. If a slightly narrow band is included, the theoretical value of around 50 lines is confirmed in one lane.
Fig. 8 shows the crossing of Azum (6) x Kanto Nakasei Gold (2) and backcrossing by Azum (6) for seven generations to search for the genes that determine the bisexuality of barley. This is a photograph comparing the lines established as two near-isogenic lines with the background of azum and the return parent of azum with the genome scanning method. A search was made for differences in 16 primer pairs in 32 lanes, but the difference between the two was extremely limited, and these were polymorphic band candidates in a region strongly linked to the bisection gene.
FIG. 9 shows an example of SCAR (Sequence Characterized Amplified Region) marker obtained by isolating a band specific to “Akitakomachi”. Primer made by inversion. B is an electrophoretic photograph showing bands amplified by PCR from 10 major varieties of nucleic acids using the primers of A. A specific band was amplified only in "Akitakomachi". control1 is a specific band isolated for sequencing as a template.
FIG. 10 shows a method for directly selecting a clone corresponding to a specific band from a genomic library using a genome scanning method. Here, a library of six genomes of blast fungus (genome size 40 MB) with an average insert size of 120 kB was divided into six sublibraries of almost one genome, and clones corresponding to the genome scanning band were separated from each sublibrary. The case of identification is shown.
In FIG. A, clones indicated by black circles are displayed as C rows, 4 columns, and a plates. A coordinate sample representing the C-th row is prepared by collecting 10 ng of the DNA of the clones in all the rows in the C-th row of all the half plates. In FIG. B, one sub-library is composed of a coordinate sample for eight rows, six columns, and six half plates, and a target genome lane, for a total of 22 lanes. Six genomes can be run on two gels at the same time, and the corresponding clone is identified from the coordinates showing the band that matches the control band.
FIG. 11 shows an example in which a genetic map of barley was prepared by electrophoresis on a 15-lane type gel, which is a precursor of the genome scanning method. Of all 272 markers, 227 AFLP markers were mapped in this manner.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.
[Example 1] Fine mapping of nucleic acid markers near the avrPib gene, a non-pathogenic gene of rice
For non-pathogenic gene avrPib of blast fungus corresponding to rice resistance gene Pi-b, we tried to search for nucleic acid markers in the vicinity and to determine the genetic distance between the obtained markers and this gene. avrPib is a non-pathogenic gene of blast fungus corresponding to the rice resistance gene Pi-b, and rice gene resistance is established by recognition of this gene product by the rice Pi-b gene, avrPib gene Mutation of the will disrupt the resistance of Pi-b. Therefore, it is necessary to isolate the gene and investigate the cause of the mutation in the strain that caused the resistance collapse and how the resistance gene product interacts with the non-pathogenic gene product. Comparison of RAPD method and RAPD method as a representative of the conventional method by crossing with blast fungus infecting Pi-b isolated from the area where resistance collapse actually occurred did.
A strain carrying avrPib (which does not infect rice with Pi-b) and a strain without avrPib (infecting rice with Pi-b) were crossed to obtain a large number of strains of the F1 generation. . After inoculating these F1 strains into rice plants having Pi-b and examining the presence or absence of avrPib in each strain, a bulk of genomic DNA was prepared from more than a dozen strains each of the avrPib holding group (+) and the deletion group (-) Then, AFLP analysis was performed with 64 × 4 = 256 primer pairs using 4 kinds of samples of 2 parent strains and 2 kinds of + and − bulks. Since the genome size of blast fungus is about 40 MB, which is about 1/10 of rice, the genome can be covered with about 1/16 of the primer pair of rice.
For comparison, the results were compared with the results of amplification using about 540 primers by the RAPD (Random Amplified Polymorphic DNAs) method. In the RAPD method, for example, 144 lanes can be run simultaneously at the same time on a large submarine gel, but in the bulk method, 540 × 4 = 2160 lanes need to be run, and it takes 15 days, Only 1860 bands were compared between the bulks, and about 100 parental polymorphism bands were found.
On the other hand, in this method, about 5700 bands could be compared and searched in 4 days, and 304 polymorphic bands were found (Table 1). This indicates that the efficiency of this method is 11 times that of the RAPD method (5700/1860 × 15/4).

Among these polymorphic bands, RAPD and AFLP showed 6 and 41 candidate near-gene bands, for which there was a clear difference between dominant homozygous and recessive homozygous by the bulk method. These bands were tested using 12 actual F1 strains, and primary screening was performed (FIG. 5). Further, the number of F1 strains was increased, and finally, an accurate map of the vicinity of avrPib was prepared by testing with 125 F1 loaf strains (FIG. 6). The RAPD method showed two bands and the present method showed 12 bands within 20 CM of the actual target gene avrPib. The band closest to the target gene was 5.3 CM in the RAPD method, whereas it was 1.8 CM in the one obtained by this method. Since 1 CM is equivalent to about 80 kB for blast fungus, this distance can be said to be a distance that can be sufficiently transferred to physical map creation.
[Example 2] Precise mapping of bc-3, a rice Camillaz mutant gene
Nucleic acid markers near the causative gene of bc-3, one of the mutants (camillaz: brittle culm) that could no longer synthesize cellulose required for secondary thickening of the rice rod cell wall, were searched by genome scanning. As a parent strain for hybridization for F2 analysis, hybridization with japonica type bc-3 mutant: M11 and indica type wild type Kasalath was used. By analyzing up to the F3 generation, 10 mutant homozygous individuals and 8 wild-type homologous individuals were identified, and a mixture of each genomic DNA was subjected to bulk analysis by the genome scanning method combined with AFLP together with the parent strains. did. After double digestion of each nucleic acid with two enzymes, EcoR I (recognition of 6 bases) and Mse I (recognition of 4 bases), combination of 1430 primers with 3 bases selected nucleotide adjacent to the cleavage site of each enzyme For, a genome scanning method by bulk analysis was applied. FIG. 7 shows an example.
As a result, 97 nearby marker candidates were obtained, of which 50 could be effectively used to detect recombinant markers from recessive homologous individuals. For each marker candidate, perform narrowing down markers by performing F2 analysis with 10 recessive homologous individuals (20 chromosomes), and in particular, as a result of analyzing 24 markers, 32 recessive homologous individuals and 64 chromosomes, the target gene and One marker was found that was co-segregated (the distance to the gene was 0 CM) (FIG. 7).
[Example 3]
In an organism having an extremely large genome size such as barley (5.5 GB), when the present invention is applied by AFLP, when the number of selected bases of the primers on both sides of the genome fragment is 3 each, rice (450 MB) Electrophoresis of double-stranded DNA under non-denaturing conditions, such as genome size, does not always result in a clear band. In this case, increasing the number of selection primers is one corresponding method.However, even if three selection markers are used, the denaturing conditions of 6 to 8.5 M urea are added to the gel, and the sample buffer is also 50%. % Of formamide and heat denaturation at 90 ° C. for 3 minutes immediately before the electrophoresis to run the DNA as a single strand, resulting in sufficient band resolution.
Fig. 8 shows the crossing of Azum (6) × Kanto Nakasei Gold (2) and backcrossing with Azum (6) for 7 generations in order to search for the genes that determine the bisexuality of barley. A line showing sex was selected, and a genome scanning method was used to compare a line established as two near-isogenic lines with the background of azum and the return parent azum. A search was made for differences in 16 primer pairs in 32 lanes, but the difference between the two was extremely limited, and these were polymorphic band candidates in a region strongly linked to the bisection gene.
Under these electrophoresis conditions, an average of about 58 bands are identified in barley. When barley AFLP was performed on a large sequencer gel, the average number of bands was about 88 (Castilioni et al. 1998 Genetics 149: 2039-2056), so about 67% of the bands were identified. Become. Where large gels can only be performed with an efficiency of about 32 lanes per gel in one day, the present invention can process 256 lanes in one day, so that 5 to 6 times the efficiency can be obtained.
[Example 4] Isolation of discrimination band of rice cultivar identified by genome scanning method and design of specific primer by sequence analysis
We developed a SCAR marker to easily identify commercial rice varieties, and tried to construct a system that would allow anyone to easily identify the variety by performing PCR in that case.
Using the genome scanning method (AFLP), bands that identify the 10 main rice cultivars on the market were searched using 55 primers. This detection takes only two days. A number of bands suitable for discriminating varieties were obtained. One of the bands, specific to "Akitakomachi", was electrophoresed in a wide lane, stained with a fluorescent dye (vistra green), cut out, and treated with TE buffer. After crushing and extracting in the PCR, it was amplified by PCR using primers for AFLP. After inserting the PCR amplification product into an appropriate plasmid and introducing it into E. coli, and culturing and amplifying the bacterium, confirm that the desired insert is contained in the resulting plasmid with restriction enzymes, analyze the sequence, and analyze the target band. A primer that specifically amplifies was designed (FIG. 9A). Using these primers, PCR amplification was performed using DNAs obtained from 10 main varieties as templates, and it was confirmed that a specific band was amplified only in “Akitakomachi” (FIG. 9B).
[Example 5] Method of selecting library clone corresponding to nucleic acid marker
Usually, when identifying a clone corresponding to a specific band obtained by the present method from a genomic library, the SCAR marker obtained as in Example 3 is used to identify a clone on a high-density membrane on which the library is mounted. Although colony hybridization is performed to select positive clones, it is too laborious and costly to use this method for a large number of bands, and the internal sequence of the isolated band is not necessarily unique. Not always.
A method for directly selecting a library clone corresponding to a specific band by genome scanning without isolating the marker band may be as follows. First, DNA of a genomic clone such as BAC is extracted from all library clones by a plasmid extractor to prepare a plate having a DNA having the same sequence as the plate of the original clone. Next, the whole genome library is divided into a set of sublibraries of a size equal to or less than one genome. This ensures that on average only one clone in a sublibrary corresponds to a particular band. Furthermore, 10 ng of DNA is collected from clones with the same coordinate number using the row, column, and plate numbers as the coordinates from several microplate clones constituting each sublibrary, and correspond to each coordinate. Make a coordinate sample. For example, from the clone in the third row, DNA is collected by collecting 10 ng of DNA irrespective of the plate number or column number, and used as a coordinate sample on the third row. Similarly, a coordinate sample is prepared for all coordinates, and the same genome scanning method is applied to each coordinate sample and the whole genomic sample as a control. To prepare coordinate sample DNA, even without extracting the DNA of all clones of the genomic library, the culture of each clone, which was increased to about 2 ml, was mixed as it was for each row, column, and plate, and used as a coordinate sample. Later, the DNA may be extracted from the mixed culture. If a band corresponding to the target band in the lane amplified from the control whole genome is found in the row, column, and plate, the number becomes the three-dimensional coordinate of the desired clone, and the clone can be picked up from the sublibrary . If a large number of clone candidates are present in the sublibrary, they should be collected and then subjected to the genome scanning method for final determination together with the control again (FIG. 10).
This method is most effective when all the bands obtained by a large number of genome scanning methods are mapped to library clones to create a contig of the whole genome, so as to cover the constituent clones of the genomic library. is there. As a result, the whole genome contig can be easily prepared by one person.
In FIG. 10A, when a genomic library of blast fungus (40 MB) (average 120 kB; equivalent to 6 genomes) is divided into 6 subgenomic libraries, each of which is equivalent to approximately 1 genome, 6 half plates (3 sheets) 2 shows a method for identifying a specific clone from one subgenomic library consisting of a single full-genome library. For 1 subgenomic library, the first row of row coordinates represents 10 μg of DNA from all clones in the first row of six half plates (6 columns x 6 half plates = 36 clones), representing the first row. It is a coordinate sample. In other words, in one subgenomic library, 8 × 6 × 6 = 288 clones of the entire subgenome library using a total of 20 coordinate samples, each representing 8, 6, and 6 coordinate coordinates representing the row, column, and plate axes, respectively Can be identified.
As shown in FIG.10B, 20 lanes using the 20 coordinate samples as a template and 2 lanes using the whole genome of the template as a template were simultaneously electrophoresed by a genome scanning method to easily obtain a control in 22 lanes. A clone corresponding to the obtained band can be identified from one subgenomic library.
Since one gel (66 lanes) can simultaneously run three subgenomic libraries, two gels can cover the entire genome and six sublibraries. That is, considering one AFLP with one primer pair, a search for clones corresponding to about 25 to 40 bands can be performed using two gels for a whole genome library equivalent to 6 genomes. Since four gels are used in the standard genome scanning method, clones corresponding to the 50-80 band obtained by two primer pairs in one run can be similarly identified from the whole genome library.
Thus, even if conservatively estimated, it is possible to map about 50-80 bands of two primer sets to clones in one electrophoresis, and about 1,200-2,000 bands to genome in 25 electrophoresis. Can be associated. Considering that the genome size of the blast fungus is 40 MB, the average density of the bands in the genome is 40 MB ÷ 1,500 lines ≒ 27 kB / line, assuming that 1,500 bands have been obtained, and an average of 4.4 lines for an average clone of 120 kB This band is considered to be obtained, and the density is too high for producing a genomic contig, considering that the degree of duplication of clones is 6 on average. As a result, if only the BAC plasmid is prepared, a contig of a whole genome library consisting of 6 genomes is completed in about one month.
If the plasmid automatic extractor can be fully used, plasmids for 18 plates can be prepared in about two weeks.
Industrial potential
By using the method and the electrophoresis apparatus of the present invention, for example, it has become possible to search, identify, and acquire nucleic acid markers for the following purposes extremely easily and with high efficiency.
(1) Development of a polymorphic marker that marks a single gene that controls important functions and traits by using a polymorphism of a nucleic acid near the gene.
In this case, it is not necessary that an organism having the target gene has a genomic map composed of existing markers.
(2) Identification and isolation of clones near the target gene in positional cloning
When trying to isolate and clone a single gene that controls important functions and traits by positional cloning based only on its chromosomal location information, search for a marker in the immediate vicinity and use BAC (bacterial artificial chromosome). ) Is provided to provide a marker for lifting a clone in the region near the gene from a genomic library such as In this case, it is not necessary for the organism having the target gene to have a map composed of existing markers.
(3) Genetic analysis, creation of high-density maps
In addition to providing a large number of nucleic acid markers for producing a genetic map of an organism with high efficiency, linkage analysis in F2 or RI strains is performed with high efficiency, and high-density maps are produced with high efficiency. This facilitates the analysis of quantitative traits (QTL: Quantitative Trait Loci) based on the contribution of multiple genes.
(4) Identification of varieties
A high efficiency search for marker bands to identify rice and other foods and seed varieties on the market. Further, the obtained discriminating band is isolated and cloned, and a primer for SCAR (Sequence Characterized Amplified Region) analysis is designed from the sequence analysis. By using such SCAR markers, it is also possible to perform the kind identification in a short time on the spot. In addition, the SCAR conversion of a nearby marker that marks a specific gene makes it easier to use as a marker in breeding and the like, and is also useful for catching BAC clones near the gene.
(5) Preparation of a contig covering the whole genome of an organism
Clones that constitute a genomic library of an organism can be joined together to easily produce a contig covering the entire genome. That is, after dividing a genomic library consisting of several genomes into sublibraries equivalent to about one genome, for a set of microplates constituting each sublibrary, its row, column, and plate are respectively x, y, Coordinates are created on the z-axis, and DNAs of all clones orthogonal to each coordinate are put together to prepare a coordinate sample and used as a template. By performing this by a genome scanning method in parallel with a normal control using whole genomic DNA as a template, a clone corresponding to each band found in a whole genome lane can be identified (FIG. 10). If bands are found at a density of about 1 band / 20-50 kB, a whole genome contig in which several clones are duplicated on average can be completed.

Claims (2)

A method for selecting a clone corresponding to a specific DNA band on a gel from a genomic DNA library,
(A) dividing a genomic DNA library of a specific organism into a plurality of sub-libraries each having a size of one genome or less of the organism;
(B) A step of assigning the row, column, and plate numbers of the sublibrary to all the clones included in each sublibrary (where the row, column, and plate are respectively represented by coordinates X, Y, and Z). Do),
(C) Clones showing a particular row of all plates (coordinate X clones), clones showing a particular column of all plates (coordinate Y clones), and all on a particular plate of one sublibrary Collecting the clones (coordinate Z clone group) from each other, separately extracting DNA as coordinate samples, and preparing genomic DNA prepared from the organism as a control ,
(D) Electrophoresis can be performed by equipping a plurality of gel plates of 10 to 30 cm square at the same time, and more than 64 nucleic acid samples can be simultaneously electrophoresed per gel plate. 1 is a 1mm thick discontinuous buffer type gel can be applied to a nucleic acid sample per lane 10 mu l, using electrophoresis device, the step of electrophoresis side by side and the DNA as a control and the DNA and coordinates sample,
(E) detecting by staining the DNA band as a coordinate sample and the DNA band as a control on the gel after electrophoresis,
(F) A band having the same degree of electrophoresis on the gel as the band of the target DNA in the control is a clone at any coordinate among the clone groups of the coordinate X clone group, the coordinate Y clone group, and the coordinate Z clone group. Determining whether or not
(G) selecting a clone corresponding to the determined three-dimensional coordinates from the sub-library. The method of claim 1 , wherein the method is performed to create a contig covering the entire genomic DNA of a particular organism.

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