JP2730670B2 - Synthetic oligonucleotides for selection of clubroot resistant Chinese cabbage - Google Patents
Synthetic oligonucleotides for selection of clubroot resistant Chinese cabbageInfo
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- JP2730670B2 JP2730670B2 JP7300454A JP30045495A JP2730670B2 JP 2730670 B2 JP2730670 B2 JP 2730670B2 JP 7300454 A JP7300454 A JP 7300454A JP 30045495 A JP30045495 A JP 30045495A JP 2730670 B2 JP2730670 B2 JP 2730670B2
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- Prior art keywords
- chinese cabbage
- clubroot
- resistant
- selection
- dna
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、根こぶ病抵抗性ハ
クサイの選抜に用いる合成オリゴヌクレオチドに関し、
詳しくは特定の塩基配列を有する合成オリゴヌクレオチ
ド並びに該合成オリゴヌクレオチドを用いるＤＮＡ分析
によってハクサイ個体を識別する方法と該識別方法によ
り育苗段階で根こぶ病抵抗性のハクサイ個体を選抜する
方法に関する。TECHNICAL FIELD The present invention relates to a synthetic oligonucleotide used for selection of clubroot-resistant Chinese cabbage,
More specifically, the present invention relates to a synthetic oligonucleotide having a specific base sequence, a method for identifying a Chinese cabbage individual by DNA analysis using the synthetic oligonucleotide, and a method for selecting a root-knot disease-resistant Chinese cabbage individual at the seedling raising stage by the identification method.
【０００２】[0002]
【従来の技術】ハクサイの根こぶ病は重要な土壌病害で
あり、一旦発生すると永年にわたって発生し、防除がき
わめて困難である。現在十分な抵抗性を持つ経済品種が
育成されていないため、殺菌剤を用いて根こぶ病の防除
を行っているが、この方法では完全に根こぶ病を回避す
ることはできない。また、殺菌剤の使用は、残留成分に
よる環境への悪影響が懸念されている。BACKGROUND OF THE INVENTION Clubroot disease of Chinese cabbage is an important soil disease, and once it occurs, it occurs for many years, and it is extremely difficult to control it. Since no economic varieties with sufficient resistance have been bred at present, fungicides are used to control clubroot, but this method cannot completely prevent clubroot. In addition, there is a concern that the use of a disinfectant may adversely affect the environment due to residual components.
【０００３】根こぶ病抵抗性の飼料用カブを素材として
利用し、根こぶ病抵抗性のハクサイの育種が行われてい
る。しかし、わが国の青果市場に適し、安定した抵抗性
を示す品種はない。この飼料用カブを素材として、わが
国の青果市場に適し、安定した抵抗性を示すハクサイの
育成を新たに試みる場合、該抵抗性素材が飼料用カブで
あるため、育種が甚だ困難であり、かつ長期にわたる。
さらに、現在の抵抗性の検定及び抵抗性個体の選抜方法
では、温度及び土壌水分が低い場合に、検定精度が低く
なるという問題点がある。このため、検定は季節的な制
約を受ける。また、検定精度を高めるためには、人工気
象室等の施設が必要となり、選抜育種の効率化を阻んで
いる。このような状況下、育苗段階で根こぶ病抵抗性を
有する個体を安定的、かつ効率的に選抜する方法の開発
が望まれていた。[0003] The root rot disease resistant Chinese cabbage has been bred by using a turnip-resistant feed turnip as a material. However, there are no varieties that are suitable for the Japanese vegetable market and show stable resistance. Using this feed turnip as a material, suitable for the vegetable market in Japan, when trying to grow a new Chinese cabbage that shows stable resistance, because the resistant material is a feed turnip, breeding is extremely difficult, and For a long time.
Furthermore, the current methods of testing resistance and selecting resistant individuals have the problem that when temperature and soil moisture are low, the accuracy of the test is reduced. For this reason, the test is subject to seasonal constraints. In addition, in order to increase the test accuracy, facilities such as artificial climate chambers are required, which hinders efficient selection and breeding. Under such circumstances, it has been desired to develop a method for stably and efficiently selecting individuals having clubroot resistance at the seedling raising stage.
【０００４】[0004]
【発明が解決しようとする課題】本発明の目的は、育苗
段階で根こぶ病抵抗性を有する個体を安定的、かつ効率
的に選抜するために、根こぶ病抵抗性ＤＮＡマーカーを
獲得し、それを用いた選抜方法を確立することである。
そこで、本発明者は任意の配列を持つ合成オリゴヌクレ
オチドを用いたポリメラーゼ連鎖反応（ＰＣＲ）によ
り、植物ＤＮＡを分析する方法を用い、根こぶ病抵抗性
ハクサイの選抜に有効なＤＮＡマーカーの獲得を試み、
検討を重ね、その獲得に成功した。このＤＮＡマーカー
を用いることにより、育種過程で根こぶ病抵抗性個体を
効率的に選抜できることを見出し、本発明を完成した。SUMMARY OF THE INVENTION An object of the present invention is to obtain a clubroot-resistant DNA marker in order to stably and efficiently select an individual having a clubroot-resistant plant at the seedling raising stage. The purpose is to establish a selection method using it.
Therefore, the present inventor used a method of analyzing plant DNA by polymerase chain reaction (PCR) using a synthetic oligonucleotide having an arbitrary sequence to obtain a DNA marker effective for selection of clubroot-resistant Chinese cabbage. Try,
After repeated examinations, we succeeded in acquiring it. By using this DNA marker, it was found that a clubroot resistant individual could be efficiently selected during the breeding process, and the present invention was completed.
【０００５】[0005]
【課題を解決するための手段】本発明は、配列表の配列
番号１〜２のいずれかに記載した塩基配列を有する合成
オリゴヌクレオチドを提供すると共に、ハクサイより抽
出したＤＮＡを鋳型とし、当該合成オリゴヌクレオチド
を用いたＰＣＲによりＤＮＡの増幅を行い、得られた増
幅産物を電気泳動分析することを特徴とする根こぶ病抵
抗性を有するハクサイの識別方法と当該識別方法により
育苗段階で根こぶ病抵抗性を有するハクサイ個体を選抜
する方法を提供するものである。SUMMARY OF THE INVENTION The present invention provides a synthetic oligonucleotide having a base sequence described in any of SEQ ID NOS: 1 and 2 in the sequence listing, and using the DNA extracted from Chinese cabbage as a template, A method for identifying Chinese cabbage having root-knot disease resistance, wherein the DNA is amplified by PCR using an oligonucleotide and the obtained amplification product is subjected to electrophoretic analysis, and the root-knot disease at the seedling raising stage by the identification method. It is intended to provide a method for selecting a Chinese cabbage individual having resistance.
【０００６】[0006]
【発明の実施の形態】以下に本発明を詳細に説明する。
任意の配列を持つ合成オリゴヌクレオチドを用いたＰＣ
Ｒにより得られるＤＮＡ多型は、ＲＡＰＤ(Random Ampl
ified Polymorphic DNA)とも言われ、用いる合成オリゴ
ヌクレオチドの種類を変えることにより、容易に、かつ
数多く得ることができ、このＤＮＡ多型を示すバンドそ
のものがＤＮＡマーカーとなり得る。この手法は、対象
とする生物より抽出されたＤＮＡを鋳型とし、ＰＣＲに
よる増幅を行い、増幅されたＤＮＡの分析により、簡単
に種々の生物におけるＤＮＡ多型を得る方法である。こ
の方法は、これまで遺伝解析や品種，個体の識別、さら
にはＦ1 種子の純度検定等に用いられてきた。DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
PC using a synthetic oligonucleotide having an arbitrary sequence
The DNA polymorphism obtained by R is RAPD (Random Ampl
It is also referred to as a conjugated polymorphic DNA), and can be easily obtained in large numbers by changing the type of the synthetic oligonucleotide used, and the band itself showing this DNA polymorphism can be a DNA marker. This method is a method in which DNA extracted from a target organism is used as a template, amplification is performed by PCR, and the amplified DNA is analyzed to easily obtain DNA polymorphisms in various organisms. This method, genetic analysis and breed far, identification of individuals, more have been used to purity test or the like of the F 1 seeds.
【０００７】本発明者は、この手法を根こぶ病抵抗性ハ
クサイ選抜に利用するため、根こぶ病抵抗性カブと罹病
性ハクサイを交雑し、その後代から抵抗性の系統を得
た。さらに、選抜に有効なＤＮＡマーカーを得るため、
交雑に用いた両系統と育成した抵抗性の系統についてＤ
ＮＡ多型を示す合成オリゴヌクレオチドの検索を行っ
た。その結果、いくつかの増幅産物（ＤＮＡバンド）
が、根こぶ病抵抗性ハクサイの選抜に有効である可能性
が示唆された。[0007] In order to use this technique for selection of clubroot-resistant Chinese cabbage, the present inventors crossed the clubroot-resistant turnip and the diseased Chinese cabbage, and obtained resistant strains from their progeny. Furthermore, to obtain an effective DNA marker for selection,
D for both lines used for crossing and resistant lines bred
A search was made for synthetic oligonucleotides exhibiting NA polymorphism. As a result, some amplification products (DNA bands)
However, it was suggested that it might be effective for selection of clubroot resistant Chinese cabbage.
【０００８】そこで、再び根こぶ病抵抗性カブと罹病性
ハクサイを交雑し、その雑種第一代植物の小胞子（未成
熟花粉）を培養し、再分化した植物の自殖系統（純系）
を用いて抵抗性と増幅産物との関係を解析し、配列表の
配列番号１〜２に示した塩基配列を有するオリゴヌクレ
オチドプライマーＲＡ１２−７５（配列番号１）及びＷ
Ｅ４９（配列番号２）による増幅産物それぞれ１本が根
こぶ病抵抗性ハクサイの選抜に有効であることを究明し
た。これらプライマーを用いて得られる増幅産物は、根
こぶ病抵抗性ハクサイを選抜するためのマーカーとなり
得る。本発明の合成オリゴヌクレオチドは、市販のＤＮ
Ａ／ＲＮＡ合成機を使用して合成することができる。[0008] Therefore, the clubroot resistant turnip and the diseased Chinese cabbage are crossed again, the microspores (immature pollen) of the first hybrid plant are cultured, and an inbred line of a regenerated plant (pure line)
Was used to analyze the relationship between the resistance and the amplification product, and oligonucleotide primers RA12-75 (SEQ ID NO: 1) having the nucleotide sequences shown in SEQ ID NOs: 1 and 2 in the sequence listing and W
It was determined that one of the amplification products obtained by E49 (SEQ ID NO: 2) was effective for selection of clubroot-resistant Chinese cabbage. An amplification product obtained using these primers can be a marker for selecting clubroot-resistant Chinese cabbage. The synthetic oligonucleotide of the present invention is commercially available DN
It can be synthesized using an A / RNA synthesizer.
【０００９】根こぶ病抵抗性を有するハクサイの識別
は、ハクサイより抽出したＤＮＡを鋳型として、上記の
合成オリゴヌクレオチドを用いたＰＣＲによってＤＮＡ
の増幅を行い、得られた増幅産物を常法に従って電気泳
動分析することによって実施することができる。このよ
うにして、根こぶ病抵抗性を有するハクサイ個体を選抜
する。The identification of Chinese cabbage having resistance to clubroot is carried out by PCR using the DNA extracted from Chinese cabbage as a template and the above-mentioned synthetic oligonucleotide.
And the obtained amplification product is subjected to electrophoretic analysis according to a conventional method. In this way, Chinese cabbage individuals having resistance to clubroot are selected.
【００１０】[0010]
【実施例】次に、本発明を実施例により詳しく説明する
が、本発明はこれらによって制限されるものではない。 実施例１ 根こぶ病抵抗性品種Ｓｉｌｏｇａ（飼料用カブ）と罹病
性品種Ｈｏｍｅｉ（ハクサイ）を交雑し、そのＦ1 の小
胞子（未成熟花粉）を培養（植物バイオテクノロジーI
I, p137, 東京化学同人に準じる。）して得られた胚様
体から再分化した植物を自殖して得られた系統を用いて
根こぶ病の検定及びＰＣＲを行った。Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited by these examples. Example 1 clubroot resistant varieties Siloga the (fodder turnip) and susceptible cultivar Homei (Chinese cabbage) crossing, the cultured microspores (immature pollen) of F 1 (Plant Biotechnology I
I, p137, according to Tokyo Chemical Dojin. ), A plant obtained by selfing a plant regenerated from the embryoid body obtained above was subjected to a test for clubroot disease and PCR.
【００１１】まず、開花前日のＨｏｍｅｉの蕾から葯を
取り除いて除雄した柱頭に、あらかじめ袋をかけて隔離
したＳｉｌｏｇａの花粉を受粉させて交雑した後、再び
袋をかけて他の花粉が交雑しないように隔離し、種子
（Ｆ1 ）が稔るまで植物体を栽培して種子（Ｆ1 ）を得
た。上記の種子（Ｆ1 ）を常法により播種し、栽培して
得られた植物体の長さ２．０〜３．５ｍｍ程度の蕾を集
めて、これを滅菌した。乳鉢に蕾とショ糖１０％を含む
初期培地（ＢＭ１０培地）を入れ、乳棒で擦りつぶして
小胞子（未成熟花粉）を遊離させた。この小胞子を孔径
６３μｍのメッシュで濾し、１２０×ｇで３分間遠心
し、上澄みを除去した沈殿物にＢＭ１０培地を添加し
た。この操作を３回繰り返した後、小胞子密度を５×１
０4 個／ｍｌに調整し、径６ｃｍの滅菌シャーレに２ｍ
ｌずつ流し込み、３２．５℃で１日間処理をした後、２
２℃，暗黒下で２週間、さらに２２℃弱光下で１週間培
養した。その結果、胚様体の形成が認められた。[0011] First, the stigma from which the anthers were removed from the buds of Homei on the day before flowering was emasculated and pollinated with Silago pollen which had been isolated in advance by bagging, and then re-bagged and crossed with another pollen. The seeds (F 1 ) were obtained by culturing the plant until the seeds (F 1 ) became fertile. Additional seeds (F 1) were seeded in a conventional manner, collect the bud having a length of about 2.0~3.5mm obtained plants are cultivated, and sterilized it. An initial medium (BM10 medium) containing buds and 10% sucrose was put in a mortar and crushed with a pestle to release microspores (immature pollen). The microspores were filtered through a mesh having a pore diameter of 63 μm, centrifuged at 120 × g for 3 minutes, and BM10 medium was added to the precipitate from which the supernatant was removed. After repeating this operation three times, the microspore density was increased to 5 × 1
0 and adjusted to 4 / ml, 2m in a sterile petri dish having a diameter 6cm
After treating at 32.5 ° C. for 1 day,
The cells were cultured at 2 ° C. in the dark for 2 weeks and further at 22 ° C. in a weak light for 1 week. As a result, formation of embryoid bodies was observed.
【００１２】形成した胚様体を、ショ糖１％及びゲラン
ガム０．５％を含む１／２濃度のＭＳ培地へ移植し、２
２℃，３０００ルクス，１６時間日長で３週間培養し
た。さらに、この胚様体をショ糖１％及び寒天０．８％
を含む１／２濃度のＭＳ培地へ移植し、２２℃，３００
０ルクス，１６時間日長で３週間培養し、再分化した芽
を伸長させ、順化し、再分化植物体を獲得した。このよ
うにして得られた再分化植物体の開花前日の柱頭に、あ
らかじめ袋をかけて隔離したＨｏｍｅｉの花粉を受粉さ
せた後、再び袋をかけて他の花粉が交雑しないように隔
離し、種子が稔るまで植物体を栽培した。[0012] The formed embryoid body was transferred to a 1/2 concentration MS medium containing 1% sucrose and 0.5% gellan gum, and
The cells were cultured at 3,000 lux at 16C for 3 weeks at 2 ° C for 3 weeks. Further, the embryoid body was sucrose 1% and agar 0.8%.
Transplanted into a 1/2 concentration MS medium containing
After culturing at 0 lux for 16 hours with a photoperiod of 3 weeks, the regenerated sprouts were elongated and acclimated to obtain regenerated plants. The stigma on the day before flowering of the regenerated plant thus obtained was pollinated with the previously isolated Homei pollen by sacking, and then sacked again to isolate the other pollen from crossing. Plants were cultivated until seeds became fertile.
【００１３】次いで、ハクサイ葉よりＤＮＡを常法、例
えばセチルトリメチルアンモニウムブロミド（ＣＴＡ
Ｂ）法［クローニングとシーケンス、p.252 (1989)、農
村文化社] により抽出した。このＤＮＡ２０ｎｇを鋳型
として、８００μＭ ｄＮＴＰｓ、１．５ｐｍｏｌ オ
リゴヌクレオチドプライマー（１種類）及び０．２ｕｎ
ｉｔ ＴｔｈＤＮＡポリメラーゼ（東洋紡製）を加え、
１０μｌの反応液を作成した。ＰＣＲ反応装置はアステ
ック社製ＰＣ−７００を用い、９４℃，３０秒の前処理
の後、熱変性９４℃，３０秒、アニーリング４０℃，２
分及び伸長反応７２℃, ３分の処理を１サイクルとし、
４５サイクル反復し、さらに７２℃，７分の温度処理を
行った。増幅産物は１μｇ／ｍｌのエチジウムブロミド
を含む１．５％アガロースゲル中で電気泳動することに
より、分析した。Next, DNA is obtained from Chinese cabbage leaves by a conventional method, for example, cetyltrimethylammonium bromide (CTA).
B) Extracted by the method [Cloning and Sequence, p.252 (1989), Rural Culture Company]. Using 20 ng of this DNA as a template, 800 μM dNTPs, 1.5 pmol oligonucleotide primer (one kind) and 0.2 un
Add it Tth DNA polymerase (manufactured by Toyobo),
10 μl of the reaction solution was prepared. The PCR reaction apparatus used was Astec's PC-700. After pretreatment at 94 ° C for 30 seconds, heat denaturation was performed at 94 ° C for 30 seconds, annealing at 40 ° C for 2 seconds.
And extension reaction at 72 ° C for 3 minutes as one cycle,
The cycle was repeated for 45 cycles, and the temperature treatment was further performed at 72 ° C. for 7 minutes. Amplification products were analyzed by electrophoresis in a 1.5% agarose gel containing 1 μg / ml ethidium bromide.
【００１４】ＰＣＲ増幅産物のうち抵抗性親Ｓｉｌｏｇ
ａに特異的なものを検索し、さらにその中で後代の抵抗
性系統に特異的なものを検索した。すなわち、合計２４
１種類のプライマーを用いて検索し、２２種類のプライ
マーによる２２個の増幅産物が抵抗性ハクサイ選抜に有
効である可能性があると判断された。さらに、Ｆ1 の小
胞子から再分化した個体を自殖して得られた系統におけ
る抵抗性の分離と、これら検索されたＰＣＲ増幅産物の
有無の分離（第１表）を検討したところ、プライマーＲ
Ａ１２−７５およびＷＥ４９による増幅産物、それぞれ
約６２０ｂｐ，約１０５０ｂｐのバンドが、抵抗性ハク
サイの選抜に有効であることが判明した。Among the PCR amplification products, the resistant parental silog
Those specific to a were searched, and among them, those specific to progeny resistant lines were searched. That is, a total of 24
A search was performed using one type of primer, and it was determined that 22 amplification products using 22 types of primers may be effective for selection of resistant Chinese cabbage. Furthermore, was examined and the resistance of the separation by the grid obtained by selfing the individuals regenerated from microspore of F 1, the separation of the presence or absence of these retrieved PCR amplification product (Table 1), primer R
The A12-75 and WE49 amplification products, about 620 bp and about 1050 bp, respectively, were found to be effective in selecting resistant Chinese cabbage.
【００１５】[0015]
【表１】 [Table 1]
【００１６】なお、表中の平均発病指数は個体ごとの病
害の発生程度を４段階の指数で示したものであり、０は
無発病を、１は僅かに発病、２は中程度の発病、３は激
しく発病を表しており、各系統毎に平均値を算出したも
のである。また、系統Ｎｏ．はＦ1 の小胞子から再分化
した個体を自殖して得られた系統の系統番号であり、＋
は図１に矢印で示したバンド（増幅産物）が増幅される
ことを、−は増幅されないことを、また？は増幅の有無
が不明なことを表す。The average disease index in the table indicates the degree of disease occurrence for each individual as a four-step index, where 0 indicates no disease, 1 indicates slight disease, 2 indicates medium disease, and 3 shows severe disease onset, and the average value was calculated for each strain. In addition, the system No. Is the system number of the resulting strains was selfed individuals were regenerated from microspore of F 1, +
Indicates that the band (amplification product) indicated by the arrow in FIG. 1 is amplified,-indicates that it is not amplified, and? Indicates that the presence or absence of amplification is unknown.
【００１７】上記の系統のうち、ＰＣＲによりこれら２
個のバンド（増幅産物）をすべて生じる系統を選抜する
と、その中に平均発病指数０．３以下の強度抵抗性系統
は６４％の割合で含まれることになり、母集団（全系
統）における強度抵抗性系統の割合３３％を明らかに上
回ることになる。さらに、この選抜された系統には母集
団に存在する強度抵抗性系統の５８％が含まれることに
なる。したがって、これらの増幅産物が根こぶ病抵抗性
ハクサイの選抜マーカーとして有効であることが示され
た。なお、片方のバンドだけでも抵抗性ハクサイの選抜
に利用することができる。Of the above lines, these two
When a line that generates all the bands (amplification products) is selected, 64% of the resistant lines with an average disease index of 0.3 or less are included in the line, and the intensity in the population (all lines) is determined. The percentage of resistant strains will clearly exceed 33%. In addition, the selected lines will include 58% of the strength resistant lines present in the population. Therefore, it was shown that these amplification products were effective as selection markers for clubroot-resistant Chinese cabbage. In addition, only one band can be used for selection of resistant Chinese cabbage.
【００１８】[0018]
【発明の効果】本発明によれば、根こぶ病抵抗性ハクサ
イの育種において、育苗段階で、しかも圃場を使用せず
に予備選抜を行うことができるため、個体選抜の効率の
向上、労力ならびに栽培面積の削減が可能である。According to the present invention, in breeding root-knot disease-resistant Chinese cabbage, preliminary selection can be carried out at the seedling raising stage and without using a field, thereby improving the efficiency of individual selection, labor and The cultivation area can be reduced.
【００１９】[0019]
配列番号：１ 配列の長さ：１２ 配列の型：核酸 鎖の数：１本鎖 トポロジー：直線状 配列の種類：他の核酸 合成ＤＮＡ 配列の特徴： 特徴を示す記号：unsure 但し、根こぶ病抵抗性ハクサ
イ選抜に有効なＤＮＡマーカーをＰＣＲにより増幅する
塩基配列 存在位置：全長 特徴を決定した方法：ＥSEQ ID NO: 1 Sequence length: 12 Sequence type: Nucleic acid Number of strands: 1 strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features: Unique symbol: unsure However, clubroot Nucleotide sequence to amplify a DNA marker effective for selection of resistant Chinese cabbage by PCR Location: Full length Method for determining characteristics: E
【００２０】配列 ５’−CATTATGCGGGC−３’Sequence 5'-CATTATGCGGGC-3 '
【００２１】 配列番号：２ 配列の長さ：１２ 配列の型：核酸 鎖の数：１本鎖 トポロジー：直線状 配列の種類：他の核酸 合成ＤＮＡ 配列の特徴 特徴を示す記号：unsure 但し、根こぶ病抵抗性ハクサ
イ選抜に有効な遺伝子マーカーをＰＣＲにより増幅する
塩基配列 存在位置：全長 特徴を決定した方法：ＥSEQ ID NO: 2 Sequence length: 12 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbols: unsure, but root Nucleotide sequence to amplify gene markers effective for selection of clubroot-resistant Chinese cabbage by PCR Location: Full length Method for determining characteristics: E
【００２２】配列 ５’−CACGTTATCGCA−３’Sequence 5'-CACGTTATCGCA-3 '
【図１】 ＰＣＲ増幅産物の電気泳動写真である。FIG. 1 is an electrophoresis photograph of a PCR amplification product.
矢印は根こぶ病抵抗性カブＳｉｌｏｇａに特異的に増幅
されるＤＮＡバンドを示す。ａはプライマーＲＡ１２−
４７（配列番号１）を、ｂはプライマーＷＥ４９（配列
番号２）をそれぞれ示す。Ｍは分子量マーカーを、Ｓは
Ｓｉｌｏｇａ（飼料用カブ）を、ＨはＨｏｍｅｉ（ハク
サイ）を示す。Arrows indicate DNA bands that are specifically amplified by clubroot resistant turnip Silloga. a is the primer RA12-
47 (SEQ ID NO: 1) and b represents primer WE49 (SEQ ID NO: 2). M indicates a molecular weight marker, S indicates Silloga (for turnip), and H indicates Homei.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 ＫＯＲＥＡＮ ＢＩＯＣＨＥＭＩＣＡ Ｌ ＪＯＵＲＮＡＬ，27（１）（1994) Ｐ．51−54 ──────────────────────────────────────────────────続 き Continuation of the front page (56) References KOREA BIOCHEMICAL L JOURNAL, 27 (1) (1994) 51-54
Claims (4)
有する合成オリゴヌクレオチド。1. A synthetic oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing.
有する合成オリゴヌクレオチド。2. A synthetic oligonucleotide having the nucleotide sequence of SEQ ID NO: 2 in the sequence listing.
し、請求項１〜２のいずれかに記載の合成オリゴヌクレ
オチドを用いたポリメラーゼ連鎖反応によりＤＮＡの増
幅を行い、得られた増幅産物を電気泳動分析することを
特徴とする根こぶ病抵抗性を有するハクサイの識別方
法。3. Amplification of DNA by polymerase chain reaction using the synthetic oligonucleotide according to any one of claims 1 to 2 using DNA extracted from Chinese cabbage as a template, and electrophoretic analysis of the resulting amplification product. A method for identifying Chinese cabbage having clubroot resistance.
病抵抗性を有するハクサイ個体を選抜する方法。4. A method for selecting Chinese cabbage individuals having clubroot resistance by the identification method according to claim 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7300454A JP2730670B2 (en) | 1995-10-26 | 1995-10-26 | Synthetic oligonucleotides for selection of clubroot resistant Chinese cabbage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7300454A JP2730670B2 (en) | 1995-10-26 | 1995-10-26 | Synthetic oligonucleotides for selection of clubroot resistant Chinese cabbage |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH09117284A JPH09117284A (en) | 1997-05-06 |
| JP2730670B2 true JP2730670B2 (en) | 1998-03-25 |
Family
ID=17884997
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7300454A Expired - Lifetime JP2730670B2 (en) | 1995-10-26 | 1995-10-26 | Synthetic oligonucleotides for selection of clubroot resistant Chinese cabbage |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2730670B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105850717A (en) * | 2016-04-20 | 2016-08-17 | 上海市农业科学院 | Method for acquiring non-heading Chinese cabbage clubroot resistance germplasm resources |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0217406D0 (en) | 2002-07-26 | 2002-09-04 | Syngenta Participations Ag | Disease resistant brassicas |
| CN105543391A (en) * | 2016-02-05 | 2016-05-04 | 北京市农林科学院 | InDel molecular marker for identifying clubroot-resistant QTL (quantitative trait locus) located on Chinese cabbage A03 chromosome and application thereof |
-
1995
- 1995-10-26 JP JP7300454A patent/JP2730670B2/en not_active Expired - Lifetime
Non-Patent Citations (1)
| Title |
|---|
| ＫＯＲＥＡＮ ＢＩＯＣＨＥＭＩＣＡＬ ＪＯＵＲＮＡＬ，２７（１）（１９９４）Ｐ．５１−５４ |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105850717A (en) * | 2016-04-20 | 2016-08-17 | 上海市农业科学院 | Method for acquiring non-heading Chinese cabbage clubroot resistance germplasm resources |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH09117284A (en) | 1997-05-06 |
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