JP7037209B2 - A method for simultaneously growing male sterile and homozygous recovery gene lines using a recovery gene linkage marker - Google Patents
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Description
本発明は、育種の技術分野に属し、具体的には、回復遺伝子連鎖マーカーによりトウガラシ雄性不稔系統及びホモ接合型回復遺伝子系統を同時に育成する方法に関する。 The present invention belongs to the technical field of breeding, and specifically relates to a method for simultaneously growing a male sterile red pepper line and a homozygous recovery gene line using a recovery gene linkage marker.
トウガラシはナス科のトウガラシ属の植物で、カプサンチンとカプサイシンが豊富なため、人気がある。現在、カプサンチンの需要は増加しており、飼料添加物、化粧品、製薬業界で広く使用されている。色素トウガラシはカプサンチンを抽出するための主な原料であり、新疆、甘粛省、内モンゴルおよび他の地域で広く普及されている。しかし、生産においては特殊用途の色素トウガラシの品種が少なく、品質にばらつきがあるため、カプサンチンの含有量が高い優良品種の雑種の育成が、トウガラシ産業の発展に欠かせない。 Capsicum is a plant belonging to the genus Capsicum of the Solanaceae family and is popular because it is rich in capsanthin and capsaicin. Currently, the demand for capsanthin is increasing and it is widely used in the feed additives, cosmetics and pharmaceutical industries. The pigment capsicum is the main raw material for extracting capsanthin and is widely used in Xinjiang, Gansu, Inner Mongolia and other regions. However, in production, there are few varieties of red pepper for special purposes and the quality varies, so it is essential to grow hybrids of excellent varieties with high capsanthin content for the development of the red pepper industry.
トウガラシは、明らかな雑種強勢を伴う一般的な他家受粉作物であり、雄性不稔三系育種技術により種子生産プロセスを簡単化し、育種コストを削減し、雑種の純度を向上させることができる。雄性不稔三系育種系において、不稔系統及び回復系統の育成は非常に重要である。トウガラシの母本不稔系統の選択に関しては、１００％の雄性不稔性を確保することに加えて、良好な結実性、高い果実数、高いカプサイシン含有量、及び強い耐性を持つ材料を選択することも必要である。色素トウガラシの回復系統には良質の材料はほとんどなく、ホモ接合型回復遺伝子を有する回復性が強い材料の育成は、最初世代雑種のリスクを減らすことができる。したがって、トウガラシ育種資源の豊富化、様々な優れた特性を備えた不稔系統及びホモ接合型回復遺伝子を備えた高品質の回復系統の育成は、トウガラシ雄性不稔三系育種作業において最優先事項となっている。 Capsicum is a common allogeneic crop with obvious heterosis, and male sterile three-line breeding techniques can simplify the seed production process, reduce breeding costs and improve hybrid purity. In the male sterile triad breeding system, the breeding of sterile strains and recovery strains is very important. Regarding the selection of the maternal sterile line of capsicum, in addition to ensuring 100% male sterility, select materials with good fruit set, high fruit count, high capsaicin content, and strong resistance. It is also necessary. There are few good quality materials in the red pepper recovery line, and growing highly recoverable materials with homozygous recovery genes can reduce the risk of first-generation hybrids. Therefore, abundance of capsicum breeding resources, breeding of sterile strains with various excellent properties and high quality recovery strains with homozygous recovery genes are top priorities in the capsicum male sterile triad breeding work. It has become.
現在、トウガラシ育種に関する３系交雑育種における不稔系統的育成は、主に、多世代の戻し交雑、自殖又は特殊なマーカー性状による材料のスクリーニングによって行われている。育成プログラムの１つは、雄性不稔源と複数のトウガラシ材料との検定交雑、戻し交雑法により雄性不稔系統を育種することである。しかしながら、この方法は、育成周期が長く、一般的に６世代以上を必要とし、遺伝資源の劣化を引き起こしやすい。別の育成プログラムは、マーカー性状として葉黄と緑変を使用し、維持系統と戻し交雑することで不稔系統を育成する。しかし、この方法は特別な材料を必要とし、育種のほとんどの材料には適していない。 Currently, sterile phylogenetic breeding in tripartite breeding for capsicum breeding is primarily performed by multigenerational backcrossing, self-fertilization or screening of materials with special marker properties. One of the breeding programs is to breed male sterile lines by a test crossing method of a male sterile source and multiple capsicum materials and a backcrossing method. However, this method has a long breeding cycle, generally requires 6 generations or more, and tends to cause deterioration of genetic resources. Another breeding program uses leaf yellow and green discoloration as marker properties and breeds sterile strains by backcrossing with maintenance strains. However, this method requires special materials and is not suitable for most breeding materials.
トウガラシ回復系統の育成方法には、主に通常の育種法及び分子マーカー補助スクリーニングが含まれる。１つの方法は、複数世代の戻し交雑又は複数世代の自殖を通じて優れた特性を達成することである。しかし、この方法は多くの人的資源と材料リソースを必要とするだけでなく、植物の劣化と商品価値の損失を引き起こす。別の方法は、緑変トウガラシ細胞質雄性不稔系統と自殖系トウガラシ父本とを交雑し、葉色マーカーに従って回復系統をスクリーニングすることである。しかし、この方法は、緑変を必要とするので、すべての育種に適しているわけではない。また、トウガラシ葯の栽培により倍加後に回復系統を栽培する方法がある。しかし、この方法は操作が難しく、厳密な技術システムと実験条件を必要とする。分子生物学の発展に伴い、分子マーカー支援育種はイネ、キャベツ、コショウで広く使用されている。トウガラシでは、回復遺伝子連鎖マーカーを使用して、トウガラシ回復系統のスクリーニングを補助することができる。この方法はスクリーニング効率が高いが、これらの研究では、回復系統補助スクリーニングに使用できる分子マーカーのみが選択されており、ホモ接合型回復遺伝子を有する個体を同定できるか否かが分かっていない。本発明は、育種目標を満たす雑種第一代を基本材料として使用することにより、ホモ接合型回復遺伝子を有する系統を育種するだけでなく、同時に雄性不稔系統を選択し、育種者の材料貯蔵量を拡大することができる。 Methods for raising red pepper recovery lines mainly include conventional breeding methods and molecular marker-assisted screening. One method is to achieve excellent properties through multiple generations of backcrossing or multiple generations of self-fertilization. However, this method not only requires a lot of human and material resources, but also causes deterioration of plants and loss of commercial value. Another method is to cross a green pepper cytoplasmic male sterile line with a self-fertilized red pepper father and screen for recovery lines according to leaf color markers. However, this method requires greening and is not suitable for all breeding. There is also a method of cultivating a recovery line after doubling by cultivating capsicum anthers. However, this method is difficult to operate and requires a rigorous technical system and experimental conditions. With the development of molecular biology, molecular marker-assisted breeding is widely used in rice, cabbage and pepper. In capsicum, recovery gene linkage markers can be used to aid in the screening of capsicum recovery strains. Although this method has high screening efficiency, these studies have selected only molecular markers that can be used for recovery line-assisted screening, and it is not known whether individuals with homozygous recovery genes can be identified. The present invention not only breeds strains having homozygous recovery genes by using F1 hybrids that meet breeding goals as basic materials, but also selects male sterile strains and stores materials for breeders. The amount can be expanded.
本発明は、回復遺伝子連鎖マーカーによりトウガラシ雄性不稔系統及びホモ接合型回復遺伝子系統を同時に育成する方法を提供することを目的とする。 It is an object of the present invention to provide a method for simultaneously growing a male sterile red pepper line and a homozygous recovery gene line using a recovery gene linkage marker.
上記目的を達成するための本発明に係るトウガラシ雄性不稔系統及びホモ接合型回復遺伝子系統を同時に育成する回復遺伝子連鎖マーカーＣａｐｓｉｃｕｍ－Ｒ－８２ＣＡＰＳであって、
前記回復遺伝子連鎖マーカーＣａｐｓｉｃｕｍ－Ｒ－８２ＣＡＰＳの配列が、
Ｆ：５’－ＴＴＣＴＣＡＴＣＡＴＡＧＣＡＴＴＧＣＴＧＴＧＣＡＡＡＣＴ－３’、
Ｒ：５’－ＣＣＡＴＣＡＧＧＣＴＴＣＧＧＴＴＡＧＴＣＡ－３’、
である。
Capsicum-R-82CAPS, a recovery gene linkage marker that simultaneously grows a male sterile red pepper line and a homozygous recovery gene line according to the present invention for achieving the above object.
The sequence of the recovery gene linkage marker Capsicum-R-82CAPS
F: 5'-TTCTCATCATCATAGCATTGCTGTGCAAACT-3',
R: 5'-CCATCAGGCTTCGGTTTAGTCA-3',
Is.
また、上記目的を達成するための本発明に係る上述の回復遺伝子連鎖マーカーＣａｐｓｉｃｕｍ－Ｒ－８２ＣＡＰＳによりトウガラシ雄性不稔系統及びホモ接合型回復遺伝子系統を同時に育成する方法であって、
トウガラシＦ２世代集団を構築するステップ（１）と、
トウガラシＦ２世代個体に対して圃場稔性同定を行うステップ（２）と、
上記連鎖マーカーによりトウガラシＦ２集団中のホモ接合型回復遺伝子を有する個体の同定を行うステップ（３）と、
ホモ接合型回復遺伝子を有する系統を育成すると同時に、新しいトウガラシ不稔系統を創造するステップ（４）と、
を含む。
Further, it is a method for simultaneously growing a male sterile red pepper line and a homozygous recovery gene line by the above-mentioned recovery gene linkage marker Capsicum-R-82CAPS according to the present invention for achieving the above object.
Step (1) to build a capsicum F2 generation group,
Step (2) of field fertility identification for capsicum F2 generation individuals,
The step (3) of identifying an individual having a homozygous recovery gene in the capsicum F2 population by the above- mentioned linkage marker , and
Step (4) to create a new red pepper sterile line at the same time as growing a line having a homozygous recovery gene,
including.
さらに、前記ステップ（１）は、
育種者の育成目標を満たすトウガラシ細胞質雄性不稔三系雑種を選択するステップと、
雑種Ｆ１を育苗し、隔離網室内で雑種Ｆ１を定植するステップと、
隔離した状態で花期に自殖させ、果実が成熟した後に種子をＦ２世代種子として収穫するステップと、
Ｆ２世代種子を育苗し、隔離した状態で個体定植を行い、個体に番号を付けるステップと、
トウガラシＦ２世代集団を構築するステップと、
を含む。
Further, the step (1) is
Steps to select a capsicum cytoplasmic male sterile hybrid that meets the breeder's breeding goals,
The step of raising seedlings of hybrid F1 and planting hybrid F1 in an isolated net room,
The step of self-fertilizing in the flowering stage in an isolated state and harvesting the seeds as F2 generation seeds after the fruits have matured,
Steps to raise F2 generation seeds, plant them in isolation, and number the individuals.
Steps to build a capsicum F2 generation group,
including.
さらに、前記ステップ（２）は、
Ｆ２個体を日光温室内で基質ウェルプレートを用いて育苗し、６葉期に隔離室内で個体定植を行うＦ２個体植栽ステップ（２－１）と、
開花期にＦ２集団に対して稔性を調査し、各株の稔性を記録し、圃場における個体に札を掛けて標識し、花期の花粉量に基づいて稔性、即ち、可稔又は不稔を同定するＦ２集団稔性圃場調査ステップ（２－２）と、
を含む。
Further, the step (2) is
F2 individual planting step (2-1) in which F2 individuals are raised in a sunlight greenhouse using a substrate well plate and planted in an isolated room at the 6-leaf stage.
The fertility of the F2 population was investigated during the flowering period, the fertility of each strain was recorded, the individual in the field was tagged and labeled, and the fertility, that is, fertile or non-fertile, was based on the amount of pollen during the flowering period. F2 population fertile field survey step (2-2) to identify fertility,
including.
さらに、前記稔性の同定では、葯表面の花粉粒子が濃密である場合、稔性は可稔であり、葯に花粉粒子がない場合、不稔である。 Further, in the above-mentioned identification of fertility, fertility is fertile when pollen particles on the anther surface are dense, and sterile when there are no pollen particles on the anther.
さらに、前記ステップ（３）は、
Ｆ２集団の開花期に、圃場稔性同定の結果に基づいて、１０個の可稔個体のＤＮＡをランダムに選択して可稔プールを構築し、１０個の不稔個体のＤＮＡをランダムに選択して不稔プールを構築し、Ｆ２個体、可稔プール、不稔プールに対してＣＴＡＢ法によりＤＮＡを抽出するステップ（３－１）と、
上記連鎖マーカーにより可稔プール及び不稔プールにおいて多型性スクリーニングを行い、分子量が８２ｂｐのバンドが１本しかない個体はホモ接合型回復遺伝子を有する個体であり選択され、他のバンド型の個体を淘汰するステップ（３－２）と、
上記連鎖マーカーによる同定結果に基づいて花期に回復遺伝子座ヘテロ接合型個体を取り除き、咲いている花に対して除去、自殖を行い、ホモ接合型回復遺伝子を有する株を構築するステップ（３－３）と、
を含む。
Further, the step (3) is
During the flowering period of the F2 population, based on the results of field fertility identification, DNA of 10 sterile individuals was randomly selected to construct a fertile pool, and DNA of 10 sterile individuals was randomly selected. Then, a sterile pool is constructed, and DNA is extracted from the F2 individual, the sterile pool, and the sterile pool by the CTAB method (3-1).
Polymorphism screening was performed in the sterile pool and the sterile pool using the above- mentioned linkage marker , and individuals having only one band having a molecular weight of 82 bp were selected as individuals having a homozygous recovery gene and were selected as individuals of other band types. Step (3-2) to weed out
Based on the identification result by the above linkage marker , the recovery locus heterozygous individual is removed at the flowering stage, the flower is removed and self-fertilized, and a strain having a homozygous recovery gene is constructed (3-). 3) and
including.
さらに、Ｆ２集団個体の数は１００－１５０株の範囲にある。 In addition, the number of F2 population individuals is in the range of 100-150 strains.
さらに、前記ステップ（４）は、
Ｆ２集団個体のうち、花期調査の結果が雄性不稔でありかつ上記連鎖マーカー同定の結果が分子量が１０８ｂｐである１本のバンドのみを有する個体は、細胞質雄性不稔個体であり、札を掛けて番号を付け、花期に育種者が所有する同じ種類のトウガラシの維持系統と検定交雑を行うステップ（４－１）と、
検定交雑後の種子を育苗、定植し、盛花期に稔性同定を行うステップ（４－２）と、
交雑後世が花期に１００％雄性不稔である場合、新たに創造された不稔系統であり、不稔率が５１％－９９％である場合、後世の不稔率１００％に達するまで繰り返して戻し交雑を行うことにより、新たに創造された不稔系統が得られ、不稔率が５０％未満である場合、対応する株を淘汰するステップ（４－３）と、
を含む。
Further, the step (4) is
Among the F2 population individuals, the individual having only one band whose flowering stage survey result is male sterility and the above chain marker identification result is 108 bp molecular weight is a cytoplasmic male sterility individual and is tagged. And the step (4-1) of performing test crossing with the maintenance line of the same kind of capsicum owned by the breeder during the flowering season.
Steps (4-2) of raising seedlings, planting seeds after crossing, and identifying fertility during the flowering season,
If the cross is 100% male sterile during the flowering season, it is a newly created sterile strain, and if the sterile rate is 51% -99%, it is repeated until the sterile rate of posterity reaches 100%. By performing backcrossing, a newly created sterile strain is obtained, and if the sterile rate is less than 50%, the corresponding strain is culled (4-3).
including.
従来技術と比べて、本発明は以下の利点及び技術的効果を有する。
１、本発明は、迅速で色素種類トウガラシ回復遺伝子座がホモ接合型である回復系統を育成し、雄性不稔系統を創造できる方法を提供することにより、多世代の戻し交雑及び多世代の自殖による退化及び育成のための交雑作業量が多い問題を解決することができる。
Compared with the prior art, the present invention has the following advantages and technical effects.
1. The present invention provides a method for rapidly breeding a recovery line in which the pigment type capsicum recovery locus is homozygous and creating a male sterile line, thereby allowing multi-generational backcrossing and multi-generational self-repairing. It is possible to solve the problem of a large amount of crossing work for degeneration and breeding due to breeding.
２、本発明は、色素トウガラシがホモ接合型回復遺伝子系統を有するか否かを快速に同定できる連鎖マーカー：（１）Ｃａｐｓｉｃｕｍ－Ｒ－８２ＣＡＰＳ（５‘－３’，Ｆ：ＴＴＣＴＣＡＴＣＡＴＡＧＣＡＴＴＧＣＴＧＴＧＣＡＡＡＣＴ，Ｒ：ＣＡＴＣＡＧＧＣＴＴＣＧＧＴＴＡＧＴＣＡ）を提供する。 2. The present invention is a chain marker that can rapidly identify whether or not the pigmented capsicum has a homozygous recovery gene lineage: (1) Capsicum-R-82CAPS (5'-3', F: TTCTCATCATAGCATTGCTGTGCAAACT, R: CATCAGGCTTCGGGTGAACTCA). )I will provide a.
３、本発明は、Ｆ２世代集団において不稔プール及び可稔プールを構築し、ホモ接合型回復遺伝子を有する個体を同定する方法を提供する。不稔プール及び可稔プールにおける連鎖分子マーカーの電気泳動のバンド型が可稔プールにおいて２本のバンドがあり、そのうちの１本の分子量が不稔プールのバンドと異なり、もう１本が不稔プールのバンドの分子量と同じである場合、ホモ接合型回復遺伝子を有するか否かを同定できるマーカーである。次いで可稔プールにおいて個体を同定した結果、分子量が８２ｂｐのバンドが１本しかなく、ホモ接合型回復遺伝子を有する個体である。 3. The present invention provides a method for constructing a sterile pool and a sterile pool in an F2 generation population and identifying an individual having a homozygous recovery gene. The band type of electrophoresis of the chain molecule marker in the sterile pool and the sterile pool has two bands in the sterile pool, one of which has a molecular weight different from that of the sterile pool and the other is sterile. If it has the same molecular weight as the band of the pool, it is a marker that can identify whether or not it has a homozygous recovery gene. Next, as a result of identifying an individual in the fertile pool, it is an individual having only one band having a molecular weight of 82 bp and having a homozygous recovery gene.
４、本発明の方法によりスクリーニングされたマーカーは、トウガラシがホモ接合型回復遺伝子系統を有するか否かを区別できるとともに、ＤＮＡレベルで雄性不稔系統個体を同定でき、環境要因の影響を低減できる。 4. The marker screened by the method of the present invention can distinguish whether or not the capsicum has a homozygous recovery gene lineage, can identify a male sterile lineage individual at the DNA level, and can reduce the influence of environmental factors. ..
５、本発明が必要とするトウガラシ育種の出発材料は入手が容易であり、自殖世代数が減少され、育種時間が短縮され、育種者のトウガラシ三系育種材料の貯蔵が迅速に充実される。 5. The starting material for red pepper breeding required by the present invention is easily available, the number of self-breeding generations is reduced, the breeding time is shortened, and the storage of the breeder's three-line red pepper breeding material is quickly enhanced. ..
以下、図面及び具体的な実施例により本発明の技術手段をさらに詳しく説明する。
〈実施例１〉
本発明は、トウガラシ雄性不稔三系雑種を出発材料とし、４０メッシュの隔離網室内でＦ２世代分離集団を構築する。開花期稔性の調査結果に基づいて、不稔プール及び可稔プールを構築し、トウガラシ回復遺伝子連鎖マーカーにより多型性スクリーニングを行う。可稔プールに対して電気泳動を行い、分子量８２ｂｐに２本のバンドがあり、そのうちの１本の分子量が不稔プールの分子量と異なり、もう１本の分子量が不稔プールの分子量と同じである場合、この回復遺伝子連鎖マーカーによりＦ２集団において回復遺伝子座がホモ接合型個体の同定を行うことができる。次いで、Ｆ２集団において、全ての可稔個体に対して分子マーカーによりスクリーニングを行い、１本のバンドのみを有しかつ分子量が不稔プールと異なる個体をトウガラシ回復遺伝子座ホモ接合型個体としてスクリーニングする。スクリーニングされた個体を自殖させた後世は、ホモ接合型回復遺伝子を有するトウガラシ回復系統である。また、分子マーカー及び開花期の表現型により不稔個体を二重同定スクリーニングし、育種者が所有する同じ種類のトウガラシ性状が良好な維持系統を父本とし、検定交雑及び戻し交雑により、新しいトウガラシ不稔系統を育成。これによって、トウガラシ雄性不稔三系法による交雑育種のために、ホモ接合型回復遺伝子を有する系統及び新しいトウガラシ不稔系統が備蓄される。
Hereinafter, the technical means of the present invention will be described in more detail with reference to the drawings and specific examples.
<Example 1>
The present invention uses a capsicum male sterile three-line hybrid as a starting material to construct an F2 generation segregation population in a 40-mesh isolation net chamber. Based on the results of the flowering fertility survey, a sterile pool and a sterile pool will be constructed, and polymorphism screening will be performed using the capsicum recovery gene linkage marker. Electrophoresis was performed on the sterile pool, and there are two bands at the molecular weight of 82 bp, one of which has a molecular weight different from that of the sterile pool and the other molecular weight of which is the same as that of the sterile pool. In some cases, this recovery gene linkage marker can identify individuals with homozygous recovery loci in the F2 population. Next, in the F2 population, all sterile individuals are screened with molecular markers, and individuals having only one band and having a molecular weight different from that of the sterile pool are screened as capsicum recovery locus homozygous individuals. .. The posterity in which the screened individuals were self-fertilized is a red pepper recovery line carrying a homozygous recovery gene. In addition, sterile individuals are double-identified and screened based on molecular markers and flowering phenotypes, and the breeder owns the same type of capsicum that has good properties. Nurture sterile strains. This will stockpile strains with homozygous recovery genes and new capsicum sterile strains for crossbreeding by the capsicum male sterility triad.
本実施例は、以下のステップを含む。
＜１ トウガラシ不稔系統及びホモ接合型回復遺伝子を有する系統を育成するために必要なトウガラシＦ２世代集団の構築＞
（１）収量、果形、カプサンチン含有量などの育種目標を満たすトウガラシ雄性不稔三系雑種を選択する。
（２）雑種を育苗棚内で育苗し、４０メッシュの隔離網室内で雑種Ｆ１を定植する。隔離したままで花期に自殖させ、果実が成熟した後、種子をＦ２世代種子として収穫する。
（３）Ｆ２世代種子を育苗棚内で育苗し、４０メッシュの隔離網室内で個体定植を行い、札を掛けて個体に番号を付ける。
（４）開花期での稔性同定のために記録する。
The present embodiment includes the following steps.
<1 Construction of a capsicum F2 generation population necessary for cultivating a capsicum sterile line and a line having a homozygous recovery gene>
(1) Select a male sterile triad hybrid of red pepper that meets breeding goals such as yield, fruit shape, and capsanthin content.
(2) Raise the hybrids in the nursery shelf and plant the hybrid F1 in a 40-mesh isolation net room. The seeds are harvested as F2 generation seeds after the fruits have matured and are self-fertilized during the flowering season while remaining isolated.
(3) F2 generation seeds are raised in a nursery shelf, individual planting is carried out in a 40-mesh isolation net room, and individual numbers are attached with tags.
(4) Record for fertility identification at the flowering stage.
＜２ トウガラシＦ２世代個体の圃場稔性同定＞
（２．１ Ｆ２個体植栽）
育苗棚内で基質ウェルプレートを用いて育苗し、６葉期に４０メッシュの隔離網室内で個体定植を行う。Ｆ２集団個体の数は１００－１５０の範囲内であればよい。以下の実施例では、Ｆ２世代個体の数は１２３である。
<2 Field fertility identification of capsicum F2 generation individuals>
(2.1 F2 individual planting)
Seedlings are raised in a nursery shelf using a substrate well plate, and individual planting is carried out in a 40-mesh isolation net room at the 6-leaf stage. The number of F2 population individuals may be in the range of 100-150. In the following examples, the number of F2 generation individuals is 123.
（２．２ Ｆ２集団稔性の圃場調査）
開花期にＦ２集団に対して稔性を調査し、各株の稔性を記録し、圃場における個体に札を掛けて標識する。花期の花粉量に基づいて稔性を同定する。葯表面の花粉粒子が濃密である場合、稔性は可稔であり、葯に花粉粒子がない場合、不稔である。
The fertility of the F2 population is investigated during the flowering period, the fertility of each strain is recorded, and the individual in the field is labeled with a tag. Identify fertility based on the amount of pollen in the flowering season. If the pollen particles on the anther surface are dense, the fertility is fertile, and if the anthers are free of pollen particles, they are sterile.
＜３ 分子マーカーによるトウガラシＦ２集団におけるホモ接合型回復遺伝子を有する個体の同定＞
（３．１ 個体の圃場稔性の調査結果に基づいてＦ２集団の不稔プール及び可稔プールを構築し、ＤＮＡを抽出する）
Ｆ２集団の開花期に、圃場稔性の調査及び稔性同定の結果に基づいて、１０個の可稔個体のＤＮＡをランダムに選択して可稔プールを構築し、１０個の不稔個体のＤＮＡをランダムに選択して不稔プールを構築する。Ｆ２個体、可稔プール、不稔プールに対してＣＴＡＢ法によりＤＮＡを抽出する。
<Identification of individuals with homozygous recovery genes in the capsicum F2 population by 3 molecular markers>
(3.1 A sterile pool and a sterile pool of the F2 population are constructed based on the results of the field fertility survey of the individual, and DNA is extracted)
During the flowering period of the F2 population, DNA of 10 fertile individuals was randomly selected to construct a fertile pool based on the results of field fertility survey and fertility identification, and 10 sterile individuals were constructed. DNA is randomly selected to construct a sterile pool. DNA is extracted from F2 individuals, sterile pools, and sterile pools by the CTAB method.
（３．２ 回復遺伝子連鎖マーカーを用いて可稔プール及び不稔プールにおいて適応性テストを行う）
回復遺伝子連鎖マーカーにより可稔プール及び不稔プールにおいて多型性スクリーニングを行った結果、Ｃａｐｓｉｃｕｍ－Ｒ－８２ＣＡＰＳマーカーは多型性を有するので（図１）、回復系統の補助スクリーニングに適用できるとともに、この２つのマーカーは、いずれも共顕性マーカーに属するので、回復遺伝子座がホモ接合型であるか否かの判断に適用できる。これによって、回復遺伝子座ホモ接合型回復系統のスクリーニングを補助することができ、回復遺伝子座がホモ接合型であるか否かの判断にかかる作業量を減少させることができる。
As a result of performing polymorphism screening in the sterile pool and the sterile pool using the recovery gene linkage marker, the Capsicum-R-82CAPS marker has polymorphism (Fig. 1), so that it can be applied to the auxiliary screening of the recovery line. Since both of these two markers belong to the coexistence marker, they can be applied to determine whether or not the recovery locus is homozygous. This can assist in screening for recovery loci homozygous recovery lines and reduce the amount of work required to determine if the recovery locus is homozygous.
マーカーはトウガラシＦ２集団で構築した可稔プール及び不稔プールが共顕性及び多型性を有して初めて、次の個体ホモ接合型回復遺伝子の同定及びスクリーニングに使用できる。つまり、マーカースクリーニングにおいて、マーカーＡＦＲＦ３ＣＡＰＳ（図１－１）のようなマーカーは、不稔プール及び可稔プールのいずれにも分子量が同じなバンドを有するので、バンド型が材料間に多型性を有さず、同定すべきトウガラシ材料集団に適用されない。マーカーＡＦＲＦ１ＣＡＰＳ（図１－２）のようなバンドを形成できないマーカーも適用できない。マーカー３３３６－ｌａｓｔ２－ＳＣＡＲ（図１－３）は、可稔プールにのみ１本のバンドがあり、不稔プールには目的バンドが現れないので、２つのプールに多型性を有するが、ホモ接合型回復遺伝子を有するか否かを判断できないため、このようなマーカーも使用できない。一方、マーカーＣａｐｓｉｃｕｍ－Ｒ－８２ＣＡＰＳ（図１－４）は、Ｆ２集団の可稔プールに２本のバンドがあり、その内の１本の分子量が不稔プールにおけるバンドと異なり、もう１本が不稔プールにおけるバンドの分子量と同じであるため、可稔プールを構築する個体からホモ接合型回復遺伝子を有する個体であるか否かを選別できるので、このようなバンド型のマーカーはホモ接合型回復遺伝子を同定するためのマーカーとして適用できる。 Markers can only be used for the identification and screening of the following individual homozygous recovery genes if the fertile and sterile pools constructed in the Capsicum F2 population are co-explicit and polymorphic. That is, in marker screening, a marker such as the marker AFRF3CAPS (FIG. 1-1) has a band having the same molecular weight in both the sterile pool and the sterile pool, so that the band type has polymorphism between materials. It does not exist and does not apply to the capsicum material population to be identified. A marker that cannot form a band, such as the marker AFRF1CAPS (Fig. 1-2), cannot be applied. Marker 3336-last2-SCAR (Fig. 1-3) has one band only in the sterile pool and no target band in the sterile pool, so it has polymorphism in the two pools but is homozygous. Such markers cannot be used because it cannot be determined whether or not they have a homozygous recovery gene. On the other hand, the marker Capsicum-R-82CAPS (Fig. 1-4) has two bands in the sterile pool of the F2 population, one of which has a molecular weight different from that in the sterile pool, and the other one. Since the molecular weight of the band in the sterile pool is the same, it is possible to select whether or not the individual has the homozygous recovery gene from the individuals that construct the sterile pool, so that such a band-type marker is homozygous. It can be applied as a marker for identifying recovery genes.
（３．３ マーカーによりトウガラシＦ２世代個体においてホモ接合型回復遺伝子を有する個体をスクリーニング、同定する）
前記マーカーＣａｐｓｉｃｕｍ－Ｒ－８２ＣＡＰＳによりＦ２世代集団において検出した結果を図２及び表３に示す。稔性同定結果と組み合わせて総合的に分析したところ、このマーカーは、回復系統のスクリーニングを補助することができる。このように、８２ｂｐバンドを有する個体のみはホモ接合型回復遺伝子を有する個体であり選択される。Ｆ２世代集団のうち、番号が３、１６、２７、３０、３１、３２、３６、３７、３９、４９、６０、６４、６７、７０、７２、８１、８８、９７、１０５、１１０、１１２、１１３、１１６、１１９、１２０、１２３である合計２６個の個体は回復遺伝子座ホモ接合型を有し、他のバンド型の個体を淘汰する。
他のトウガラシ材料で構築された集団では、同定作業量を減少させるために、このマーカーの適用性を改めてテストし、つまり、図１－４のバンド型を有することを確定した後、回復遺伝子座ホモ接合型個体の同定を行う必要がある。
The results detected in the F2 generation population by the marker Capsicum-R-82CAPS are shown in FIGS. 2 and 3. When analyzed comprehensively in combination with fertility identification results, this marker can assist in screening recovery lines. Thus, only individuals with the 82 bp band are individuals with homozygous recovery genes and are selected. Among the F2 generation groups, the numbers are 3, 16, 27, 30, 31, 32, 36, 37, 39, 49, 60, 64, 67, 70, 72, 81, 88, 97, 105, 110, 112, A total of 26 individuals, 113, 116, 119, 120, 123, have a recovery locus homozygous type and weed out other band type individuals.
In populations constructed with other capsicum materials, the applicability of this marker was retested to reduce the amount of identification work, ie, after confirming that it had the band type shown in Figure 1-4, the recovery locus. It is necessary to identify homozygous individuals.
（３．４ 自殖によるホモ接合型回復遺伝子を有する株の構築）
隔離網室内で、分子マーカーによる同定結果に基づいて花期に回復遺伝子座ヘテロ接合型個体を取り除き、咲いている花に対して除去、自殖を行い、ホモ接合型回復遺伝子を有する株を構築する。次いで、育種目標及び経済性に応じて、ホモ接合型回復遺伝子を有する優れた回復系統を育成する。
(3.4 Construction of a strain having a homozygous recovery gene by self-fertilization)
In the isolation net chamber, the recovery locus heterozygous individual is removed at the flowering stage based on the identification result by the molecular marker, and the flower is removed and self-fertilized to construct a strain having the homozygous recovery gene. .. Then, depending on the breeding goal and economic efficiency, an excellent recovery line having a homozygous recovery gene is cultivated.
＜４ ホモ接合型回復遺伝子を有する系統を育成すると同時に新しいトウガラシ雄性不稔系統を製造する＞
（４．１）
Ｆ２集団個体のうち、花期の表現型が無花粉であり、かつ分子マーカー同定により分子量が１０８ｂｐである１本のバンドのみを有する個体は細胞質雄性不稔個体である。分子マーカー同定結果と圃場同定結果の組み合わせにより、Ｆ２集団中の不稔株をより正確に同定でき、環境の変化により植株が不稔になることにより圃場稔性同定が正確ではないことを防止できる。Ｆ２集団における番号が８、９、１４、１７、２０、２３、２４、３３、４４、４８、５２、６１、６２、６８、６９、７６、８２、８３、８６、８９、９２、９８、９９、１０２、１０３、１０４、１０８、１１１、１２１である個体は雄性不稔個体である。札を掛けて番号を付け、花期に育種者が所有する同じ種類のトウガラシの維持系統と検定交雑を行い、記録する。
<4 Produce a new male sterile line of red pepper at the same time as growing a line having a homozygous recovery gene>
(4.1)
Among F2 population individuals, individuals having a pollen-free flowering stage phenotype and having only one band having a molecular weight of 108 bp by molecular marker identification are cytoplasmic male sterile individuals. The combination of the molecular marker identification result and the field identification result can identify the sterile strain in the F2 population more accurately, and can prevent the field fertility identification from being inaccurate due to the sterile planting due to the change in the environment. .. Numbers in the F2 population are 8, 9, 14, 17, 20, 23, 24, 33, 44, 48, 52, 61, 62, 68, 69, 76, 82, 83, 86, 89, 92, 98, 99. , 102, 103, 104, 108, 111, 121 are male sterile individuals. Tag and number, perform test crosses with maintenance strains of the same type of capsicum owned by the breeder during the flowering season and record.
（４．２）
交雑した後の種子を育苗、定植し、盛花期に稔性同定を行う。
（４．２．１）
交雑後世が花期に１００％雄性不稔である場合、新たに創造された不稔系統である。
（４．２．２）
雄性不稔率が５１％－９９％である場合、後世の不稔率１００％に達するまで繰り返して戻し交雑を行うことにより、新たに創造された不稔系統を得る。
（４．２．３）
雄性不稔率が５０％未満である場合、対応する株を淘汰する。
(4.2)
After crossing, seedlings are raised and planted, and fertility is identified during the flowering season.
(4.2.1)
If the post-crossing is 100% male sterile during the flowering season, it is a newly created sterile strain.
(4.2.2)
When the male sterility rate is 51% -99%, a newly created sterile line is obtained by repeated backcrossing until the sterility rate of posterity reaches 100%.
(4.2.3)
If the male sterility rate is less than 50%, the corresponding strain is culled.
（４．３）
新たに創造されたトウガラシ雄性不稔系統を母本とし、既存の同じ種類のトウガラシの回復系統を父本として交雑の組み合わせを作り、母本不稔系統の組合わせ能力を評価する。
(4.3)
Based on the newly created male sterility line of Pepper, and using the recovery line of the same type of existing Pepper as the father, a hybrid combination is made and the combination ability of the sterile mother line is evaluated.
以上の実施例は、本発明の技術内容を説明するためのものに過ぎず、本発明の保護範囲を制限するものではない。上記の各実施例を参照しながら本発明を詳しく説明したが、当業者であれば、本発明の技術内容の趣旨及び範囲から逸脱しない限り、上記各実施例に記載の技術内容を修正し、又は一部若しくは全部の技術内容に同等の変更を行うことができる。 The above-mentioned examples are merely for explaining the technical contents of the present invention, and do not limit the scope of protection of the present invention. The present invention has been described in detail with reference to each of the above embodiments, but those skilled in the art will modify the technical contents described in the above embodiments as long as they do not deviate from the purpose and scope of the technical contents of the present invention. Alternatively, equivalent changes can be made to some or all of the technical content.
Claims (4)
トウガラシ雄性不稔三系雑種を出発材料とし、トウガラシＦ２世代集団を構築するステップ（１）と、
トウガラシＦ２世代個体に対して圃場稔性同定を行うステップ（２）と、
前記連鎖マーカーによりトウガラシＦ２集団中のホモ接合型回復遺伝子を有する個体の同定を行うステップ（３）と、
ホモ接合型回復遺伝子を有する系統を育成すると同時に、新しいトウガラシ不稔系統を創造するステップ（４）と、
を含み、
前記ステップ（３）は、
Ｆ２世代集団の開花期に、圃場稔性同定の結果に基づいて、１０個の可稔個体のＤＮＡをランダムに選択して可稔プールを構築し、１０個の不稔個体のＤＮＡをランダムに選択して不稔プールを構築し、Ｆ２世代個体、可稔プール、不稔プールに対してＣＴＡＢ法によりＤＮＡを抽出するステップ（３－１）と、
前記連鎖マーカーにより可稔プール及び不稔プールにおいて多型性スクリーニングを行い、分子量が８２ｂｐのバンドが１本しかない個体はホモ接合型回復遺伝子を有する個体であり選択され、他のバンド型の個体を淘汰するステップ（３－２）と、
前記連鎖マーカーによる同定結果に基づいて花期に回復遺伝子座ヘテロ接合型個体を取り除き、咲いている花に対して除去、自殖を行い、ホモ接合型回復遺伝子を有する株を構築するステップ（３－３）と、
を含み、
前記ステップ（４）は、
Ｆ２世代個体のうち、花期調査の結果が雄性不稔でありかつ前記連鎖マーカー同定の結果が分子量が１０８ｂｐである１本のバンドのみを有する個体は、細胞質雄性不稔個体であり、札を掛けて番号を付け、花期に育種者が所有する同じ種類のトウガラシの維持系統と検定交雑を行うステップ（４－１）と、
検定交雑後の種子を育苗、定植し、盛花期に稔性同定を行うステップ（４－２）と、
交雑後世が花期に１００％雄性不稔である場合、新たに創造された不稔系統であり、不稔率が５１％－９９％である場合、後世の不稔率１００％に達するまで繰り返して戻し交雑を行うことにより、新たに創造された不稔系統が得られ、不稔率が５０％未満である場合、対応する株を淘汰するステップ（４－３）と、
を含み、
Ｆ２世代個体の数は１００－１５０株の範囲にある
ことを特徴とする、育成方法。 Pepper male sterility by the recovery gene linkage marker Capsicum-R-82CAPS using a primer set consisting of a primer consisting of F: 5'-TTCTCATCATAG CATTGCTGTGCAAACT-3' and a primer consisting of R: 5'-CCATCAGGCTTCGGTAGTCA-3'. It is a method of simultaneously growing a line and a homozygous recovery gene line.
Steps (1) to build a capsicum F2 generation population using capsicum male sterile three-line hybrids as a starting material,
Step (2) of field fertility identification for capsicum F2 generation individuals,
The step (3) of identifying an individual having a homozygous recovery gene in the capsicum F2 population by the linkage marker , and
Step (4) to create a new red pepper sterile line at the same time as growing a line having a homozygous recovery gene,
Including
The step (3) is
During the flowering period of the F2 generation population, based on the results of field fertility identification, DNA of 10 sterile individuals was randomly selected to construct a fertile pool, and DNA of 10 sterile individuals was randomly selected. The step (3-1) of selecting and constructing a sterile pool and extracting DNA from the F2 generation individual, the sterile pool, and the sterile pool by the CTAB method,
Polymorphism screening was performed in the sterile pool and the sterile pool using the above- mentioned linkage marker , and the individual having only one band having a molecular weight of 82 bp was selected as an individual having a homozygous recovery gene and was selected as an individual of another band type. Step (3-2) to weed out
Based on the identification result by the linkage marker , the recovery locus heterozygous individual is removed at the flowering stage, the flower is removed and self-fertilized, and a strain having a homozygous recovery gene is constructed (3-). 3) and
Including
The step (4) is
Among the F2 generation individuals, the individual having only one band whose flowering stage survey result is male sterility and the result of the chain marker identification is 108 bp in molecular weight is a cytoplasmic male sterility individual and is tagged. And the step (4-1) of performing test crossing with the maintenance line of the same kind of capsicum owned by the breeder during the flowering season.
Steps (4-2) of raising seedlings, planting seeds after crossing, and identifying fertility during the flowering season,
If the cross is 100% male sterile during the flowering season, it is a newly created sterile strain, and if the sterile rate is 51% -99%, it is repeated until the sterile rate of posterity reaches 100%. By performing backcrossing, a newly created sterile strain is obtained, and if the sterile rate is less than 50%, the corresponding strain is culled (4-3).
Including
A breeding method characterized in that the number of F2 generation individuals is in the range of 100-150 strains.
育種者の育成目標を満たすトウガラシ細胞質雄性不稔三系雑種を選択するステップと、
雑種Ｆ１を育苗し、隔離網室内で雑種Ｆ１を定植するステップと、
隔離した状態で花期に自殖させ、果実が成熟した後に種子をＦ２世代種子として収穫するステップと、
Ｆ２世代種子を育苗し、隔離した状態で個体定植を行い、個体に番号を付けるステップと、
トウガラシＦ２世代集団を構築するステップと、
を含むことを特徴とする、請求項１に記載の育成方法。 The step (1) is
Steps to select a capsicum cytoplasmic male sterile hybrid that meets the breeder's breeding goals,
The step of raising seedlings of hybrid F1 and planting hybrid F1 in an isolated net room,
The step of self-fertilizing in the flowering stage in an isolated state and harvesting the seeds as F2 generation seeds after the fruits have matured,
Steps to raise F2 generation seeds, plant them in isolation, and number the individuals.
Steps to build a capsicum F2 generation group,
The breeding method according to claim 1, further comprising.
Ｆ２世代個体を日光温室内で基質ウェルプレートを用いて育苗し、６葉期に隔離室内で個体定植を行うＦ２世代個体植栽ステップ（２－１）と、
開花期にＦ２世代集団に対して稔性を調査し、各株の稔性を記録し、圃場における個体に札を掛けて標識し、花期の花粉量に基づいて稔性、即ち、可稔又は不稔を同定するＦ２集団稔性圃場調査ステップ（２－２）と、
を含むことを特徴とする、請求項１に記載の育成方法。 The step (2) is
The F2 generation individual planting step (2-1), in which F2 generation individuals are raised in a sunlight greenhouse using a substrate well plate and planted in an isolated room at the 6-leaf stage,
During the flowering period, the fertility of the F2 generation population is investigated, the fertility of each strain is recorded, the individual in the field is tagged and labeled, and the fertility, that is, fertile or fertile or based on the amount of pollen in the flowering period. F2 population fertile field survey step (2-2) to identify sterility and
The breeding method according to claim 1, further comprising.
The identification of fertility according to claim 3, wherein the fertility is fertile when the pollen particles on the anther surface are dense, and sterile when the anthers are free of pollen particles. Training method.
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