Method for breeding new transgenic insect-resistant high-quality high-coat cotton strain
阅读说明：本技术 一种转基因抗虫优质高衣分棉花新品系的培育方法 (Method for breeding new transgenic insect-resistant high-quality high-coat cotton strain ) 是由 钟文娟 牟方生 陈正杰 陈四维 周永航 戢沛城 杨泽湖 蒋理 石盛佳 宛永璐 毛 于 2020-12-18 设计创作，主要内容包括：本发明提供了一种转基因抗虫优质高衣分棉花新品系的培育方法,涉及作物新品种选育技术领域。本发明利用转基因抗虫棉花品系与转基因高衣分品系杂交,再以杂交后代中具有转基因高衣分基因的植株为母本,以转基因抗虫优质品系为轮回亲本进行连续六代回交,筛选具有高衣分基因、抗虫的植株自交两代,再筛选具有高衣分基因、抗虫的株行进行自交繁殖,评价筛选纤维品质与所述轮回亲本基本一致的株行,即得所述转基因抗虫优质高衣分棉花新品系。本发明将常规育种方法和现代分子生物学方法有机结合,实现了基因的精准选择,减少新品系创制过程中的盲目性,缩短了育种周期。(The invention provides a method for breeding a new transgenic insect-resistant high-quality high-coat cotton strain, and relates to the technical field of breeding new crop varieties. The invention utilizes the cross breeding of a transgenic insect-resistant cotton line and a transgenic high-coat-rate line, then uses a plant with a transgenic high-coat-rate gene in a cross progeny as a female parent and uses a transgenic insect-resistant high-quality line as a recurrent parent to carry out continuous six-generation backcross, screens the plant with the high-coat-rate gene and the insect resistance for two generations of selfing, screens plant rows with the high-coat-rate gene and the insect resistance for selfing propagation, evaluates and screens plant rows with basically consistent fiber quality with the recurrent parent, thus obtaining the transgenic insect-resistant high-quality high-coat-rate cotton new line. The invention organically combines the conventional breeding method and the modern molecular biology method, realizes the accurate selection of genes, reduces the blindness in the process of creating new strains and shortens the breeding period.)
1. A method for breeding a new transgenic insect-resistant high-quality cotton strain with high coat content is characterized by comprising the following steps: (1) hybridizing the transgenic insect-resistant high-quality cotton line serving as a female parent and the transgenic high-coat line serving as a male parent to obtain a transgenic insect-resistant hybrid combination F1；
(2) Using the transgenic insect-resistant hybrid combination F1As female parent, hybridizing with the transgenic insect-resistant high-quality product line as male parent to obtain backcross first generation BC1F1；
(3) Taking a plant with a transgenic high coat-dividing gene in the backcross progeny as a female parent, taking the transgenic insect-resistant high-quality cotton product as a recurrent parent, carrying out backcross, and continuously backcrossing for five generations to obtain backcross six-generation BC6F1；
(4) For the backcross six generations BC6F1Selfing the plant with transgenic high coat gene, and harvesting the single plant to obtain BC6F2；
(5) Screening the BC6F2Selfing the plants with transgenic high coat gene in the insect-resistant plant rows with all the plants to be insect-resistant, and harvesting the single plant to obtain BC6F3；
(6) Screening the BC6F3All the plants in the strain are insect-resistant and have transgenic high-coat-length genes, and the plant rows are subjected to selfing propagation, and the plant rows are harvested, evaluated and screened to obtain the transgenic insect-resistant high-quality high-coat-length cotton new strain.
2. The method of claim 1, wherein the female parent in step (1) comprises Jinluo Chang No. 1; the male parent comprises IF 1-1.
3. The breeding method as claimed in claim 1, wherein the transgenic high coat material IF1-1 contains FBP7: iaaM gene.
4. The culture method of claim 3, wherein the PCR identification primers for iaaM include IF-up, IF-dn1 and IF-dn 2; and the nucleotide sequence of the IF-up is shown as SEQ ID NO. 1; the nucleotide sequence of the IF-dn1 is shown as SEQ ID NO.2, and the nucleotide sequence of the IF-dn2 is shown as SEQ ID NO. 3.
5. The method of claim 4, wherein the PCR-based assay system is 25 μ L, and comprises: 5 XPrimeSTAR buffer 5U L, dNTP 2U L, upstream primer and downstream primer each 1U L, cotton gDNA 10 ~ 200ng, PrimeSTAR DNApolymerase 0.5U and the balance of water.
6. A method of cultivation as claimed in claim 4 or 5, wherein the PCR identification procedure comprises: denaturation at 98 ℃ for 10sec, annealing at 56 ℃ for 5sec, and elongation at 72 ℃ for 60sec, with 32 cycles.
7. The method of claim 1, wherein the screening method for insect resistance in steps (1) to (5) comprises: and in the 5-7 leaf stage of the cotton seedling, smearing the leaf which is unfolded with a 0.8 mass percent karamycin sulfate solution, and if the leaf is not discolored, identifying that the plant has insect resistance, and if the leaf is discolored, identifying that the plant does not have insect resistance.
Technical Field
The invention belongs to the technical field of new crop variety breeding, and particularly relates to a method for breeding a new transgenic insect-resistant high-quality high-coat cotton variety.
Background
Cotton is the first economic crop in China and plays an important role in national economy, cotton fiber is an important textile raw material, and the demand for high-quality cotton fiber is increasing along with the rapid development of the economy in China and the improvement of the living standard of people.
The yield and the fiber quality of cotton belong to quantitative traits, the quantitative traits are closely related to each other in the expression heredity, the yield (clothes) of the variety with extremely high fiber quality is low, the difficulty in synchronously improving the yield and the quality of cotton fibers by traditional breeding is high, and the period is long. The university in southwest introduced FBP7 (iaaM) into Ji-Cotton 14 of cotton variety to obtain transgenic cotton material IF1-1(FBP7:: iaaM) with synchronously improved fiber yield and fineness: the number of mature fibers is increased, and the micronaire value is also obviously reduced, so that the mature fibers are listed as a new 2 nd-generation transgenic germplasm material of cotton. However, except for high clothes score, the IF1-1 has other economic properties such as boll forming property, single boll seed cotton weight and the like, and is difficult to directly popularize and apply in a large area.
The combination of transgenic biotechnology and traditional breeding technology has made great progress in the aspect of cotton genetic improvement, China utilizes the insect-resistant gene cry1Ab/cry1Ac which is independently constructed and adopts the traditional technologies of hybridization, backcross and the like to breed a large number of transgenic insect-resistant cotton varieties through a large number of cotton breeders in China, and as the insect-resistant and genetic stability of the cotton varieties are good, the use of pesticides is greatly reduced, the yield of seed cotton is obviously improved, the economic benefit and the environmental benefit are obviously improved, and the transgenic crop with the largest planting area in China is formed by large-area popularization and application in Yangtze river basin and yellow river basin for more than 20 years. The combination of transgenic biotechnology and traditional breeding technology has become one of the most effective approaches for crop breeding at present.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for breeding a new transgenic insect-resistant high-quality high-coat cotton strain, which can be used for assisting the molecular detection of genes in conventional breeding selection, thereby effectively aggregating insect-resistant, high-quality and high-coat cotton strains and shortening the breeding period.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for breeding a new transgenic insect-resistant high-quality cotton strain with high coat content, which comprises the following steps: (1) hybridizing the transgenic insect-resistant high-quality cotton line serving as a female parent and the transgenic high-coat line serving as a male parent to obtain a transgenic insect-resistant hybrid combination F1；
(2) Using the transgenic insect-resistant hybrid combination F1As female parent, hybridizing with the transgenic insect-resistant high-quality product line as male parent to obtain backcross first generation BC1F1；
(3) Taking a plant with a transgenic high coat-dividing gene in the backcross progeny as a female parent, taking the transgenic insect-resistant high-quality cotton product as a recurrent parent, carrying out backcross, and continuously backcrossing for five generations to obtain backcross six-generation BC6F1；
(4) For the backcross six generations BC6F1Selfing the plant with transgenic high coat gene, and harvesting the single plant to obtain BC6F2；
(5) Screening the BC6F2Selfing the plants with transgenic high coat gene in the insect-resistant plant rows with all the plants to be insect-resistant, and harvesting the single plant to obtain BC6F3；
(6) Screening the BC6F3All the plants in the strain are insect-resistant and have transgenic high-coat-length genes, and the plant rows are subjected to selfing propagation, and the plant rows are harvested, evaluated and screened to obtain the transgenic insect-resistant high-quality high-coat-length cotton new strain.
Preferably, the female parent in step (1) comprises Jinluchong No. 1; the male parent comprises IF 1-1.
Preferably, the transgenic high-coat material IF1-1 contains FBP7: iaaM gene.
Preferably, the PCR identification primer of the high-coatinggene FBP7, iaaM comprises IF-up, IF-dn1 and IF-dn 2; and the nucleotide sequence of the IF-up is shown as SEQ ID NO. 1; the nucleotide sequence of the IF-dn1 is shown as SEQ ID NO.2, and the nucleotide sequence of the IF-dn2 is shown as SEQ ID NO. 3.
Preferably, the PCR-identified system is 25 μ L, and comprises: 5 XPrimeSTAR buffer 5U L, dNTP 2U L, upstream primer and downstream primer each 1U L, cotton gDNA 10 ~ 200ng, PrimeSTAR DNApolymerase 0.5U and the balance of water.
Preferably, the PCR identification procedure comprises: denaturation at 98 ℃ for 10sec, annealing at 56 ℃ for 5sec, and elongation at 72 ℃ for 60sec, with 32 cycles.
Preferably, the method for screening for pest resistance in steps (1) to (5) comprises: and in the 5-7 leaf stage of the cotton seedling, smearing the leaf which is unfolded with a 0.8 mass percent karamycin sulfate solution, and if the leaf is not discolored, identifying that the plant has insect resistance, and if the leaf is discolored, identifying that the plant does not have insect resistance.
The invention provides a method for breeding a transgenic insect-resistant high-quality high-coat cotton new strain, which comprises the steps of hybridizing a transgenic insect-resistant high-quality cotton strain with a transgenic high-coat cotton strain, carrying out continuous six-generation backcross by taking a plant with a transgenic high-coat gene in filial generations as a female parent and the transgenic insect-resistant high-quality strain as a recurrent parent, screening plants with the transgenic high-coat gene and insect resistance for two generations of selfing, screening plant rows with the high-coat gene and insect resistance for selfing propagation, evaluating and screening plant rows with the fiber quality basically consistent with that of the recurrent parent, and obtaining the transgenic insect-resistant high-quality high-coat cotton new strain. The breeding method organically combines a conventional breeding method and a molecular identification method, has reasonable steps and simple operation, reduces the blindness of creating new strains, shortens the breeding period and realizes accurate breeding.
Drawings
FIG. 1 is a flow chart of the cultivation of a new transgenic insect-resistant high-quality cotton strain with high coat content;
FIG. 2 shows the result of PCR detection of the molecular marker for detecting whether the material contains transgenic high-coat genes.
Detailed Description
The invention provides a method for breeding a new transgenic insect-resistant high-quality cotton strain with high coat content, the process of which is shown in figure 1 and comprises the following steps: (1) hybridizing the transgenic insect-resistant high-quality cotton line serving as a female parent and the transgenic high-coat line serving as a male parent to obtain a transgenic insect-resistant hybrid combination F1；
(2) Using the transgenic insect-resistant hybrid combination F1As female parent, hybridizing with the transgenic insect-resistant high-quality product line as male parent to obtain backcross first generation BC1F1；
(3) Taking a plant with a transgenic high coat-dividing gene in the backcross progeny as a female parent, taking the transgenic insect-resistant high-quality cotton product as a recurrent parent, carrying out backcross, and continuously backcrossing for five generations to obtain backcross six-generation BC6F1；
(4) For the backcross six generations BC6F1Selfing the plant with transgenic high coat gene, and harvesting the single plant to obtain BC6F2；
(5) Screening the BC6F2Selfing the plants with transgenic high coat gene in the insect-resistant plant rows with all the plants to be insect-resistant, and harvesting the single plant to obtain BC6F3；
(6) Screening the BC6F3All the plants are insect-resistant and have transgenic high coat gene, and the plant lines are selfed and propagatedAnd harvesting, evaluating and screening to obtain the transgenic insect-resistant high-quality high-coat-rate cotton new strain.
The invention takes a transgenic insect-resistant cotton line as a female parent and takes a transgenic high-clothes-division line as a male parent for hybridization to obtain a transgenic insect-resistant hybrid combination F1. The female parent preferably comprises Jinlugao No.1 (approved variety of Sichuan province in 2009, approved number is Chuanjuan cotton 2009006, single boll seed cotton is 6.1g in weight, lower in clothing score: 35.22%, and particularly excellent in fiber quality, the average length of the upper half part of the fiber is 35.42mm, the specific strength is 35.9cN/tex, and the micronaire value is 3.92), and the male parent preferably comprises IF1-1 (introduced from southwest university, the clothing score of the fiber is 45.7%, but the boll is smaller, and the weight of the single boll seed cotton is 4.7g, and is disclosed in Liu Jiu, Jian high-clothing cotton germplasm IF1-1, and the application of heterosis and breeding, Hebei agricultural science, 2016, 20 (3): 70-74). In the present invention, the high coat gene in the transgenic high coat line is preferably FBP7:: iaaM, and the primers for detecting the gene preferably include IF-up (SEQ ID NO.1, 5'-atcagagccatgaataggtc-3'), IF-dn1(SEQ ID NO.2, 5'-ccttgcagaccagtaagggc-3'), and IF-dn2(SEQ ID NO.3, 5'-gtgtgataccccaaattggg-3'). In the present invention, when the gene is identified, the PCR system is preferably 25 μ L, and includes: 5 XPrimeSTAR buffer (Mg)2+Plus) 5. mu. L, dNTP (2.5 mM each) 2. mu.L, 1. mu.L each of the forward and reverse primers (10 mM each), 10-200 ng of cotton gDNA, 0.5U of PrimeSTAR DNApolymerase, and balance water. The PCR program of the present invention is preferably 98 ℃ denaturation 10sec, 56 ℃ annealing 5sec, 72 ℃ extension 60sec, run 32 cycles. According to the present invention, it is preferable that 10. mu.L of PCR product is electrophoresed after amplification, and IF-up + IF-dn1DNA band (690bp) and IF-up + IF-dn2DNA band (883bp) are simultaneously amplified, so that the detected plant contains the target gene (FIG. 2). In the subsequent steps, the detection and screening methods for the genes are the same, and thus are not described in detail later.
In the present invention, before the hybridization, the screening of the female parent for insect resistance is preferably further included, and the screening preferably includes: and in the 5-7 leaf stage of the cotton seedling, smearing the leaf which is unfolded with a 0.8 mass percent karamycin sulfate solution, and if the leaf is not discolored, identifying that the plant has insect resistance, and if the leaf is discolored, identifying that the plant does not have insect resistance. The screening methods for insect resistance are the same, so the subsequent content is not repeated. The crossing of the invention can lead the transgene high coat-dividing gene FBP7 to be that iaaM and insect-resistant gene are polymerized in the same offspring.
Obtaining transgenic insect-resistant hybrid combination F1Then, the invention uses the transgenic insect-resistant hybrid combination F1As female parent, hybridizing with the transgenic insect-resistant high-quality product line as male parent to obtain backcross first generation BC1F1. The male parent of the invention is preferably the same as the transgenic insect-resistant high-quality line in the above step. The invention relates to a backcross which can transfer half of genetic components of recurrent parent to filial generation BC1F1。
Get backcross generation BC1F1Then, the invention takes the plant with the transgene high coat-dividing gene in the backcross offspring as the female parent and takes the transgene insect-resistant high-quality cotton product line as the recurrent parent for continuous backcross for five generations to obtain the backcross six generations BC6F1. The co-backcrossing of six generations as described herein delivers almost all of the genetic components of the recurrent parent to the BC6F1In (1), make BC6F1The main genetic components of (A) are basically consistent with the recurrent parent.
Get backcross six generations BC6F1Then, the invention carries out backcross on the BC of six generations6F1Selfing the plant with transgenic high coat gene, and harvesting the single plant to obtain BC6F2. The plant which is homozygous for the insect-resistant gene and has high coat-dividing gene can be obtained by selfing.
Get BC6F2Then, the present invention screens the BC6F2Selfing the plants with transgenic high-coat genes in the insect-resistant plant rows, and harvesting single plants to obtain BC6F3. The plant homozygous for the insect-resistant gene and the high-coat-rate gene can be obtained by selfing.
Get BC6F3Then, the present invention is directed to the BC6F3Selfing the plant line with transgenic high coat gene, harvesting, evaluating, screening the plant line with retained quality basically same as that of recurrent parentAnd obtaining the transgenic insect-resistant high-quality high-coat-rate cotton new strain. The selfing of the invention can combine high-quality genes and combine the genes respectively homozygous for insect-resistant, high-quality and high-clothes-mark genes, and simultaneously, the genes and the expressed characters thereof can be stably inherited to the next generation.
In the invention, according to the Mendelian crop genetic law, the cross can gather partial genetic backgrounds among different individuals on the same individual, the backcross can increase the genetic background of recurrent parents into hybrid offspring, and theoretically, the genetic components of the recurrent parents contained in the hybrid offspring are increased by half every 1 backcross. Through 5-6 times of continuous backcross, the main characters of the offspring are close to those of recurrent parents, namely the material formed by six backcross generations basically keeps the required existing excellent characters. In general, the gene to be screened is at BC6F1The gene is heterozygous, the heterozygous gene can be homozygous through selfing, the homozygous gene can be obtained through continuous 2-3 generations of selfing selection, and the expressed characters can be stably inherited to the next generation.
The method for breeding a new transgenic insect-resistant high-quality cotton line with high coat length is described in detail below with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Breeding of new transgenic insect-resistant high-quality high-coat cotton strain RFB
(1) In summer 2015, transgenic insect-resistant high-quality variety Jinluzhangong No.1 and transgenic high-coat material IF1-1 (introduced from southwest university and containing FBP7:: iaaM gene) were planted in the adults. When 7 true leaves are completely unfolded after the Jinluhong No.1 grows, smearing the unfolded leaves with a 1.0 mass percent solution of the kalamycin sulfate, the leaves of all plants are not discolored after 9 days, the Jinluhong No.1 resists 100 percent of insects, and the insect-resistant genes are homozygous; when 6 true leaves are completely unfolded after IF1-1 grows, the high coat-dividing gene FBP7 is identified by PCR for each plant, iaaM, and 690bp DNA band and 883bp DNA band are simultaneously amplified by each plant, which shows that IF1-1 is homozygous. After the flowering period, the stamens of the flowers on the Jinluhong No.1 plant are removed, the flowers of IF1-1 are used for pollinating the flowers for hybridization, and 5 seeds of hybrid bolls are harvested to obtain the transgenic insect-resistant genic male sterile hybrid F1 (material name RF).
(2) In 2015 winter, planting hybrid combination RF and transgenic insect-resistant high-quality variety Jinluzhangsu No.1 in Hainan, after blooming, removing stamen by RF, pollinating with flower of Jinluzhangsu No.1, and hybridizing to obtain hybridized boll seed to obtain backcross first generation material (material name RFBC)1F1)。
(3)2016 in summer, planting backcross generation material RFBC in the growth period1F1And Jinluhong No.1, to RFBC in 7 true leaf stages1F1The 20 strains are transformed into FBP7, the iaaM gene is identified by PCR, 12 strains of plants without FBP7 and 8 strains of plants with FBP7 and the iaaM gene are eliminated and reserved. In the flowering period, 8 retained flowers are pollinated and hybridized by using Jinluzhangong No.1, and seeds of 16 hybrid bells are harvested to obtain backcross second-generation material seeds (material name RFBC)2F1)。
By the same method, the Jinluong No.1 is taken as a recurrent male parent, a plant containing FBP7, iaaM gene in a backcross material is taken as a female parent, and backcross is continuously carried out for five generations to obtain a backcross six-generation material RFBC6F1。
(4) Planting RFBC in Hainan in 2018 winter6F1Obtaining 22 plants, carrying out FBP7 (iaaM identification) when 7 true leaves are low to obtain 10 transgenic high coat plants, carrying out flower-tying selfing, harvesting single plants, and harvesting 5 selfing bolls for each plant to obtain RFBC6F2Seeds;
(5) planting RFBC in the summer of 20196F2The material comprises 10 plant rows, wherein 15 plants are planted in each plant row, the insect resistance is identified by using 1% of the carramycin sulfate when 6 true leaves are planted, and 4 plants grow out of existing plants which are not insect-resistant and are eliminated; all the plants in the other 6 plant rows are insect-resistant, and before the bud stage, each plant is respectively subjected to FBP7, iaaM identification, 33 plants which are not high-coat gene-divided plants are eliminated, robust, insect-resistant and high-coat gene-divided plants 30 are selected, flower-tying selfing is carried out, each plant is independently harvested, 5 selfing bells are respectively harvested from each plant, and the FBC is obtained6F3And (4) seeds.
(6) In 2019, 30 harvested in summer are planted in HainanAn FBC6F3The materials are planted separately to form 30 plant rows, and each plant row contains 15 plants. When 5 true leaves are used for carrying out insect resistance identification and FBP7:: iaaM PCR identification, all plants are insect-resistant, 22 plant rows have plants without FBP7:: iaaM, and the plants are eliminated. All the plants in the remaining 8 plant rows are insect-resistant and have high coat-dividing genes, are bred, are respectively mixed for harvest, the coat, the fiber quality, the boll weight, the boll-forming property and the like of each plant row are considered and evaluated, and 5 plant rows which are obviously improved compared with the No.1 coat of Jinluo and have the equivalent fiber quality, boll weight and boll-forming property are screened out: RFB-1, RFB-5, RFB-13, RFB-21, RFB-27, namely obtaining the transgenic insect-resistant high-quality high-coat cotton new strain (RFB).
TABLE 1 major traits of transgenic insect-resistant high-coat new lines RFB
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> institute for breeding and cultivating economic crops of academy of agricultural sciences of Sichuan province
<120> cultivation method of new transgenic insect-resistant high-quality high-coat-rate cotton strain
<141> 2020-12-17
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atcagagcca tgaataggtc 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccttgcagac cagtaagggc 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gtgtgatacc ccaaattggg 20
- 上一篇：一种医用注射器针头装配设备
- 下一篇：一种三蒴型芝麻的高产定向改良方法