JP5721960B2 - Primer set for discriminating the origin of Yokoshihikari and method for discriminating the origin of rice - Google Patents
Primer set for discriminating the origin of Yokoshihikari and method for discriminating the origin of rice Download PDFInfo
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Description
本発明は、イネコシヒカリの産地判別用プライマーセット及びイネコシヒカリの産地判別方法に関する。 [0001] The present invention relates to a primer set for discriminating the origin of rice plant and a method for discriminating the origin of rice plant.
従来より、コシヒカリと他の品種を区別する方法はあったが（例えば特許文献１参照）、コシヒカリ品種内の産地間区別を行われていない。 Conventionally, there has been a method of distinguishing Koshihikari from other varieties (see, for example, Patent Document 1), but no distinction has been made between production areas in Koshihikari varieties.
全国で栽培されているコシヒカリは、その品種としての信憑性が社会的な問題となっている。同時に、コシヒカリには産地間差があることが知られているが、それを厳密に分類する方法はこれまで開発されていない。
本発明の目的は、新潟産のコシヒカリとそれ以外のコシヒカリとを判別することができるイネコシヒカリの産地判別用プライマーセット及びイネコシヒカリの産地判別方法を提供することにある。 An object of the present invention is to provide a primer set for discriminating the production area of rice koshihikari and a method for discriminating the production area of rice koshihikari, which can discriminate Koshihikari from Niigata and other koshihikari.
本発明のイネコシヒカリの産地判別用プライマーセットは、配列番号１で示される塩基配列を有する第１プライマーと、配列番号２で示される塩基配列を有する第２プライマーとを具備する。
本発明のこのような構成のプライマーセットを用いてＰＣＲを行うことにより、新潟産のコシヒカリとそれ以外のコシヒカリとを判別することができる。
The primer set for discriminating the origin of the rice field of the present invention comprises a first primer having a base sequence represented by SEQ ID NO: 1 and a second primer having a base sequence represented by SEQ ID NO: 2.
By performing PCR using the primer set having such a configuration of the present invention, it is possible to distinguish between Koshihikari from Niigata and other Koshihikari.
本発明のイネコシヒカリの産地判別方法は、上述に記載のプライマーセットとイネコシヒカリから抽出したＤＮＡを用いてＰＣＲを行い、前記ＰＣＲで得られた産物を電気泳動する。 In the method for discriminating the origin of the rice cultivar of the present invention, PCR is carried out using the primer set described above and the DNA extracted from the rice cultivar, and the product obtained by the PCR is electrophoresed.
すなわち、イネゲノムの複数の部位から、コシヒカリと他の品種を区別するＤＮＡ断片を見いだし、さらにコシヒカリ品種内の違いを検出するＤＮＡ断片により、産地間の区別を行う。ＤＮＡ断片の検出はＰＣＲ法により実施する。 That is, DNA fragments that distinguish Koshihikari from other varieties are found from a plurality of parts of the rice genome, and further, production regions are distinguished by DNA fragments that detect differences in Koshihikari varieties. Detection of DNA fragments is performed by PCR.
イネのトランスポゾンを指標にして、系統特異的なＤＮＡ断片を探索した結果、コシヒカリを区別することのできる複数のＤＮＡマーカーを得、さらに新潟タイプのコシヒカリとそれ以外のコシヒカリ（福井タイプのコシヒカリ）を判別するＤＮＡマーカーを取得した。 As a result of searching for strain-specific DNA fragments using rice transposon as an index, we obtained multiple DNA markers that can distinguish Koshihikari. In addition, Niigata-type Koshihikari and other Koshihikari (Fukui-type Koshihikari) A DNA marker for discrimination was obtained.
本発明によるＤＮＡマーカー群は、高精度にコシヒカリを他の品種と区別することができ、コシヒカリ品種内の産地間の違い（新潟タイプと福井タイプ）を区別することができた。 The DNA marker group according to the present invention was able to distinguish Koshihikari from other varieties with high accuracy, and was able to distinguish the difference (Niigata type and Fukui type) between the production areas within the Koshihikari cultivar.
本発明のこのような構成によれば、新潟産のイネコシヒカリを試料としたＰＣＲ産物のみに所定の増幅が確認されるため、増幅が確認できれば試料が新潟産のイネコシヒカリであると判別でき、増幅が確認できなければ試料が新潟産のイネコシヒカリでないことを判別することができる。 According to such a configuration of the present invention, since the predetermined amplification is confirmed only in the PCR product using Niigata rice cultivated as a sample, if the amplification can be confirmed, it can be determined that the sample is Niigata cultivated rice. If the amplification cannot be confirmed, it can be determined that the sample is not Niigata rice cultivar.
以下、本発明の実施形態を説明する。
（プライマーセット）
Embodiments of the present invention will be described below.
(Primer set)
本実施形態におけるイネコシヒカリの産地判別用プライマーセットは、配列番号１で示される塩基配列を有する第１プライマー（primerL）と、配列番号２で示される塩基配列を有する第２プライマー（primerR）とを具備する。 The primer set for discriminating the origin of rice koshihikari in the present embodiment includes a first primer (primerL) having the base sequence represented by SEQ ID NO: 1 and a second primer (primerR) having the base sequence represented by SEQ ID NO: 2. It has.
プライマー設計においては、イネゲノムの配列から、新潟産のコシヒカリとそれ以外のコシヒカリとを判別するための標的として、配列番号３に示されるの遺伝子がコードされている領域（染色体８番の位置にある）を選んだ。
配列番号３に示される遺伝子に関する付加的情報は以下のとおりである。
chr8 64561〜65265
gene Os08t0101000-01
product 新潟274 bp(福井mPing-in 704 bp)
（ＰＣＲを用いた判別方法）
試料として、新潟産のコシヒカリと福井産のコシヒカリとを使用した。
＜DNAの抽出＞
１）イネサンプルの葉をハサミで1〜2mmに刻み、1.5mlエッペンドルフチューブに入れる。
２）サンプルの入ったチューブを液体窒素の入った容器に浸してサンプルを凍結させる（2〜3分間）。
３）ホモジナイザーでサンプルを粉砕する（解凍しないよう素早く）。
粉砕されたサンプルに抽出用バッファーを400μl加え、ボルテックスミキサーを用いて懸濁する。室温に15分以上放置する（時々攪拌）。
４）等量(400μl)のクロロホルム／イソアミルアルコール（24：1）を加え、激しく振って混合する。
５）13000rpmで10分間遠心する（室温）。
６）マイクロピペットを使い、上清を別のチューブに移す（300μl程でよい）。
７）等量の2-プロパノールを加え、転倒操作により混和する。
８）13000rpmで10分間遠心する（室温）。９）チューブの底にペレット状のDNAを確認したら、上清をデカントで捨てる。
１０）70%エタノールを1ml加え、軽く撹拌してDNAペレットを洗う。
１１）13000rpmで5分間遠心する（室温）。
１２）マイクロピペットでエタノールを出来る限り除去した後、DNAペレットをデシケータ中で5分間減圧乾固する。
１３）50μlのTEバッファーを加えてDNAを溶解し、DNAサンプルとした。
以上のプロトコルにより各ＤＮＡを抽出し溶液とした。
＜電気泳動＞
In the primer design, as a target for discriminating Koshihikari from Niigata and other Koshihikari from the sequence of the rice genome, the region encoded by the gene shown in SEQ ID NO: 3 (located at chromosome 8) ) Was selected.
Additional information regarding the gene shown in SEQ ID NO: 3 is as follows.
chr8 64561〜65265
gene Os08t0101000-01
product Niigata 274 bp (Fukui mPing-in 704 bp)
(Determination method using PCR)
As samples, Koshihikari from Niigata and Koshihikari from Fukui were used.
<DNA extraction>
1) Cut rice sample leaves into 1-2mm with scissors and place in 1.5ml Eppendorf tube.
2) Immerse the tube containing the sample in a container containing liquid nitrogen to freeze the sample (2-3 minutes).
3) Crush the sample with a homogenizer (quickly not to thaw).
Add 400 μl of extraction buffer to the ground sample and suspend using a vortex mixer. Leave at room temperature for more than 15 minutes (sometimes stirring).
4) Add an equal volume (400 μl) of chloroform / isoamyl alcohol (24: 1) and shake vigorously to mix.
5) Centrifuge at 13000 rpm for 10 minutes (room temperature).
6) Using a micropipette, transfer the supernatant to another tube (about 300 μl is enough).
7) Add an equal volume of 2-propanol and mix by inverting.
8) Centrifuge at 13000 rpm for 10 minutes (room temperature). 9) After confirming the pelleted DNA at the bottom of the tube, decant the supernatant.
10) Add 1 ml of 70% ethanol and gently stir to wash the DNA pellet.
11) Centrifuge at 13000 rpm for 5 minutes (room temperature).
12) After removing ethanol as much as possible with a micropipette, the DNA pellet is dried under reduced pressure in a desiccator for 5 minutes.
13) DNA was dissolved by adding 50 μl of TE buffer to obtain a DNA sample.
Each DNA was extracted by the above protocol to prepare a solution.
<Electrophoresis>
PCR反応液は1μMのプライマー、2.5mMの塩化マグネシウム、PCRバッファー、250μMのd-NTPおよび0.5μlのTaqポリメラーゼにDNA0.5μgを加え調製した。PCRの反応条件は変性温度94℃(30秒)、アニーリング温度をプライマーのTm値にあわせ(30秒)、伸長反応72℃(PCR産物の長さに応じて30秒より1kb)において行い、35サイクルの増幅を実施した。 The PCR reaction solution was prepared by adding 0.5 μg of DNA to 1 μM primer, 2.5 mM magnesium chloride, PCR buffer, 250 μM d-NTP and 0.5 μl Taq polymerase. PCR reaction conditions were a denaturation temperature of 94 ° C (30 seconds), an annealing temperature adjusted to the Tm value of the primer (30 seconds), an extension reaction at 72 ° C (from 30 seconds to 1 kb depending on the length of the PCR product), 35 A cycle amplification was performed.
その結果、図１左側に示すように、新潟産のコシヒカリについてのみ２７４ｂｐの位置にバンドが認められた。尚、図１右側は福井産のコシヒカリのＰＣＲ産物を電気泳動の結果である。 As a result, as shown on the left side of FIG. 1, a band was observed at a position of 274 bp only for Koshihikari from Niigata. The right side of FIG. 1 shows the results of electrophoresis of Koshihikari PCR products from Fukui.
以上のように、上述のプライマーセットを用いてＰＣＲを行った後、電気泳動を行うことによって、その電気泳動パターンから、試料のコシヒカリが新潟産であるか否かを検出することができる。すなわち、産地が不明なコシヒカリがある場合、試料のコシヒカリから抽出したＤＮＡが上述のプライマーセットを用いてＰＣＲによって２７４ｂｐのバンドが増幅されるのであれば新潟産と判別でき、704 bpのバンドが増幅されるのであれば新潟産ではないと判別できる。 As described above, after performing PCR using the above primer set, it is possible to detect whether or not Koshihikari of the sample is from Niigata from the electrophoresis pattern by performing electrophoresis. That is, if there is Koshihikari whose origin is unknown, it can be distinguished from Niigata if the DNA extracted from Koshihikari of the sample amplifies a 274 bp band by PCR using the above primer set, and a 704 bp band is amplified. If it is, it can be determined that it is not from Niigata.
Claims (4)
配列番号２で示される塩基配列を有する第２プライマーと
を具備するイネコシヒカリの産地判別用プライマーセット。 A first primer having a base sequence represented by SEQ ID NO: 1,
A primer set for discriminating the origin of rice cultivar, comprising a second primer having the base sequence represented by SEQ ID NO: 2.
前記プライマーセットとイネコシヒカリから抽出したＤＮＡを用いたＰＣＲで得られる産物の電気泳動において、イネコシヒカリが新潟産であれば２７４ｂｐのバンドを増幅させ、イネコシヒカリが新潟産でなければ７０４ｂｐのバンドを増幅させるIn electrophoresis of a product obtained by PCR using the primer set and DNA extracted from Yoneshihikari, a 274 bp band was amplified if Yoneshihikari was from Niigata, and a 704 bp band was obtained if Yoneshihikari was not from Niigata. Amplify
プライマーセット。Primer set.
前記電気泳動によって２７４ｂｐのバンドが増幅されれば新潟産と判別し、７０４ｂｐのバンドが増幅されれば新潟産ではないと判別するIf the 274 bp band is amplified by the electrophoresis, it is determined that the product is from Niigata, and if the 704 bp band is amplified, it is determined that the product is not from Niigata.
イネコシヒカリの産地判別方法。A method for discriminating the origin of the rice plant.
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