Method for constructing point mutation mapping group by using germ cold shock double-induction method
The cotton mutation mapping population was constructed through the radiation-induced germ cold shock double-induced germ, which solved the problems of long population construction cycle and limited material utilization in the study of cotton fiber quality, achieved efficient and fast progress in mutant screening and breeding, and promoted the improvement of cotton quality and disease and pest resistance.
Patent Information
- Application Number
- CN202510602587.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-12
- Publication Date
- 2025-08-12
AI Technical Summary
In the prior art, there is a lack of effective and fast research groups in the study of cotton fiber quality, resulting in low competitiveness of the cotton industry, and the construction cycle of traditional mapping groups is long and the use of materials is limited, making it difficult to meet the needs of fast and efficient breeding.
The radiation-induced germ cold shock double-induced induced method was used to perform high-energy electron linear acceleration irradiation using a 10MeV/20kW anti-wave electron accelerator, combined with gibberellin GA3 treatment, and performed step-by-step mutagenesis treatment and resequencing screening to construct point mutation mapping populations, including the primary mutagenesis M0 generation and the secondary mutagenesis M0 generation, and the target traits were screened generation by generation to form stable mutants.
It has achieved rapid construction of efficient cotton mutation mapping groups, increased mutation efficiency by more than 40%, shortened cycle, provided rich polymorphic sites, promoted the breeding of cotton fiber quality and disease resistance, reduced the use of chemical fertilizers, and comply with environmentally friendly policies.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of crop research mapping population construction, and particularly relates to a method for constructing a point mutation mapping population using a germ cold shock double inducement method. Background Art
[0002] my country is the world's leading cotton producer, with cotton growing belts distributed across Xinjiang, Henan, Jiangsu, Hubei, Shandong, Hebei, and Anhui. Regionally, there are three major cotton-producing regions: the Xinjiang cotton belt, the Yellow River Basin cotton belt, and the Yangtze River Basin cotton belt. Xinjiang cotton, primarily encompassing Xinjiang and Gansu, is the primary cotton source for domestic consumption. Cotton quality has become a key factor in industrial competitiveness, both domestically and internationally. According to a survey by the China Cotton Textile Association, market demand for mid- to high-end, high-quality cotton accounts for approximately 70% of the total cotton market. National demand is estimated to be over 3 million tons, of which Xinjiang accounts for over 2 million tons, representing 50-60% of Xinjiang's total cotton production. However, despite declining overall production in recent years, my country's raw cotton inventory remains high, with textile companies importing large quantities of cotton from the United States and Australia. This is primarily due to the low quality of raw cotton produced in my country and rising production costs, which have led to the industry's struggle for market competitiveness. Although the sequencing of the cotton genome was completed in 2015, providing key genetic information references for molecular breeding and the creation of new cotton varieties, in actual research and breeding processes, key genetic information on cotton fiber quality is still lacking, and the resolution of excellent varieties is insufficient. The main reason is the lack of a strong, effective and fast research group, which seriously hinders the further improvement of the quality of the bred varieties.
[0003] Research populations can be divided into two categories based on their genetic stability: temporary populations, such as F2, F3, F4, BC, and tri-cross populations. In these populations, the segregating unit is the individual. Once selfed or inbred, their genetic composition changes, making them unusable permanently. Permanent segregating populations, such as RIL and DH populations, are the segregating unit in these populations. While genotypic differences exist between lines, individuals within a line are identical and homozygous, demonstrating self-insegregation. These populations can reproduce offspring through selfing or inbreeding without altering their genetic composition, making them permanently usable. DNA linkage maps can be constructed using different types of segregating populations. To date, mapping population construction has primarily relied on naturally derived materials, constructed through hybridization or backcrossing. These methods are not only limited by the availability of available materials but also require lengthy construction cycles. Once constructed, they often suffer from irreparable defects, limiting their potential for reuse. These shortcomings of genetically segregating mapping populations make them inadequate for current rapid and efficient research strategies. A rapid, efficient, and practical method for constructing mapping populations is urgently needed.
[0004] Irradiation mutagenesis uses high-energy radiation to alter the genetic material of an organism, resulting in different trait variations between the first and subsequent generations. Two common methods of crop mutagenesis are physical and chemical. Physical mutagenesis primarily involves radiation, such as electron beams, lasers, X-rays, gamma rays, and cosmic rays. Chemical mutagenesis is also a common method used in breeding research. Commonly used chemical mutagens include alkylating agents, azides, base analogs, and nitroso compounds. Each mutagenesis method has its own unique characteristics, advantages, and disadvantages.
[0005] Electron accelerators have the advantages of high emission power, high radiation utilization rate, short irradiation processing time, adjustable energy, uniform absorbed dose, simple safety protection requirements, no need to regularly replenish the irradiation source, no residual nuclear waste pollution, wide application range, low investment and low operating cost. In addition, high-energy electron linear acceleration irradiation mutagenesis technology has the advantages of multiple-point mutations, sufficient variation, wide genome coverage, moderate mutation, high mutagenesis efficiency and fast material homozygosity.
[0006] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention
[0007] The object of the present invention is to provide a method for constructing a point mutation mapping population, which utilizes radiation-induced point mutations as polymorphic sites generated in crop genomes to form a new method for constructing point mutation mapping populations. The method is simple and efficient, has rich sites, and a simple and fast construction process, which can save time, manpower and material resources. It is easy to promote and can obtain batches of cotton mutants in a short period of time, construct mutation mapping populations for various single traits and multiple traits, and provide material support for scientific research.
[0008] To achieve the above object, the present invention provides the following technical solutions:
[0009] A method for constructing a point mutation mapping population using a germ cold shock double-induction method comprises the following steps:
[0010] (1) Select conventional cotton varieties that are currently widely used in agricultural production and have been accurately sequenced;
[0011] (2) selecting high-quality dry seeds from the cotton varieties of step (1), subjecting them to mutagenesis treatment, and obtaining primary mutagenized M0 generation seeds;
[0012] (3) The primary mutagenized M0 generation seeds were soaked in gibberellin GA3 at 25°C for 6 h, then fished out and subjected to secondary mutagenization treatment after cold fluorescent irradiation at 18°C for 12 h to obtain secondary mutagenized M0 generation seeds;
[0013] (4) Sowing the secondary mutagenesis M0 generation seeds, taking young leaves at the three-leaf and one-heart stage, resequencing them against the sequencing reference genome, performing point mutation detection, screening individual plants with target traits, and harvesting mutant M1 generation seeds;
[0014] (5) From all seeds of the M1 generation harvested from single sowing, young leaves at the three-leaf, one-heart stage were taken and resequenced against the sequencing reference genome for point mutation detection and mutant M2 seeds were harvested from individual plants;
[0015] (6) From all seeds of the M2 generation harvested from single sowing, young leaves at the three-leaf, one-heart stage were taken and resequenced against the sequencing reference genome for point mutation detection and mutant M3 seeds were harvested from individual plants;
[0016] (7) All seeds of the M3 generation harvested from single seed sowing were screened for extreme cases of the target trait, and young leaves at the three-leaf, one-heart stage were taken and resequenced against the sequencing reference genome for point mutation detection;
[0017] (8) Based on the specific situation of point mutation detection, rationally screen the mutation mapping population.
[0018] Preferably, the cotton variety in step (1) is Zhongmian 113 or Zhongmian 24.
[0019] Preferably, the mutagenic treatment in step (2) and step (3) is carried out by electron beam, laser, X-ray, gamma ray, cosmic ray or high-energy electron linear acceleration irradiation, or by EMS, alkylating agent, azide compound, base analogue or nitroso compound treatment.
[0020] Preferably, the mutagenesis treatment in step (2) is high-energy electron linear acceleration irradiation, with an irradiation dose of 350 Gy and an irradiation time of 2 s.
[0021] Preferably, the secondary mutagenesis treatment in step (3) is high-energy electron linear acceleration irradiation, with an irradiation dose of 150 Gy and an irradiation time of 2 s.
[0022] Preferably, the concentration of gibberellin GA3 in step (3) is 120 ppm.
[0023] Preferably, the target traits screened in step (4) include leaf shape, size and color, plant height, seed size, cotton fiber length, strength, micronaire value and lint percentage.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] (1) The present invention adopts a 10MeV / 20kW reverse wave electron accelerator Packaged irradiated cotton seeds were subjected to a step-by-step induction strategy to improve induction efficiency. A dose of 250 Gy was used for mutagenesis, resulting in a secondary M0 generation of seeds. Planting material was then selected for target traits, including leaf shape, size, and color, plant height, kernel size, and cotton fiber length, strength, micronaire value, and lint percentage. A point mutation mapping population was constructed using fiber quality traits. The mutagenesis was effective, with significant etiolation mutations in the first generation and moderate lethality. The results were stable over two to three generations of continuous planting, making this method highly efficient and effective for screening phenotypic cotton mutants and a valuable tool for selecting new, specialized, and high-quality cotton materials.
[0026] (2) The present invention constructs a new point mutation mapping population, which can effectively break the cumbersome procedures of general population construction. It has the advantages of short cycle, great effect, simple design, reusability, human control, advanced means and is conducive to breaking the cumbersome linkage. It fundamentally solves the unfavorable factors that polymorphic sites are controlled by factors such as genetic exchange, breaks the dilemma of the lack of effective mapping populations in cotton fiber quality research, accelerates the pace of research on new fiber quality genes, and will greatly promote the breeding of cotton germplasm that improves cotton fiber quality and combines high yield, disease resistance and insect resistance, highlighting high quality, reducing the use of chemical fertilizers and pesticides, and is conducive to the implementation of environmentally friendly policies.
[0027] (3) The present invention utilizes radiation-induced point mutations as polymorphic sites generated in crop genomes to form a new method for constructing point mutation mapping populations. The method is simple and efficient, has rich sites, and a simple and fast construction process. It can save time, manpower and material resources, and is easy to promote. It can obtain batches of cotton mutants in a short period of time, construct mutation mapping populations for various single traits and multiple traits, and provide material support for scientific research. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 These are normal seedlings of Zhongmian 113 without mutation;
[0029] Figure 2 These are the mutant seedlings of Zhongmian 113 obtained after irradiation mutagenesis;
[0030] Figure 3 This is the fiber length mutation mapping population for Zhongmian 113;
[0031] Figure 4 This is the carding diagram of part of the material in the group of fiber length mutation mapping for Zhongmian 113. DETAILED DESCRIPTION
[0032] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts are within the scope of protection of the present invention.
[0033] In this specification, the machine used for irradiation mutagenesis treatment is a 10MeV / 20kW reverse wave electron accelerator
[0034]
[0035] Example
[0036] A method for constructing a point mutation mapping population using a germ cold shock double-induction method comprises the following steps:
[0037] (1) Select Zhongmian 113, a conventional cotton variety currently used in agricultural production and accurately sequenced;
[0038] (2) selecting high-quality dry seeds from the cotton varieties obtained in step (1) and sealing them in cartons, with each carton weighing 5 kg, and irradiating the packaged cotton seeds with high-energy electron linear acceleration at a dose of 350 Gy and an irradiation time of 2 s to induce irradiation, thereby obtaining primary mutagenized M0 generation seeds;
[0039] (3) soaking the primary mutagenized M0 generation seeds in 120 ppm gibberellin GA3 at 25°C for 6 h, then taking them out and irradiating them with cold fluorescent light at 18°C for 12 h for secondary mutagenization treatment. The mutagenization treatment method was the same as step (2) to obtain secondary mutagenized M0 generation seeds;
[0040] (4) The secondary mutagenesis M0 generation seeds were rinsed, soaked overnight, and sown. Young leaves at the three-leaf and one-heart stage were taken and resequenced against the sequencing reference genome for point mutation detection. Individual plants with the target trait of cotton fiber length were screened, and mutant M1 seeds were harvested.
[0041] (5) From all seeds of the M1 generation harvested from single sowing, young leaves at the three-leaf, one-heart stage were taken and resequenced against the sequencing reference genome for point mutation detection and mutant M2 seeds were harvested from individual plants;
[0042] (6) From all seeds of the M2 generation harvested from single sowing, young leaves at the three-leaf, one-heart stage were taken and resequenced against the sequencing reference genome for point mutation detection and mutant M3 seeds were harvested from individual plants;
[0043] (7) All seeds of the M3 generation harvested from single seed sowing were screened for extreme cases of the target trait, and young leaves at the three-leaf, one-heart stage were taken and resequenced against the sequencing reference genome for point mutation detection;
[0044] (8) Based on the specific situation of point mutation detection, rationally screen the mutation mapping population.
[0045] Figure 1 These are normal seedlings of Zhongmian 113 without mutation, and were self-pollinated to maintain purity.
[0046] Figure 2 These are the mutant seedlings of Zhongmian 113 obtained after irradiation mutagenesis;
[0047] Its advantages are as follows: through the double mutagenesis strategy of two irradiation mutagenesis (350Gy+150Gy combined with gibberellin cold shock (120ppm GA3, 18℃ cold fluorescence irradiation), the mutation efficiency is significantly improved. After the first mutagenesis, gibberellin is used to break the seed dormancy, cold shock enhances the embryo sensitivity and performs appropriate cold shock repair. The second mutagenesis further induces multiple point mutations, breaking the limitations of traditional single mutagenesis. Continuous single seed sowing from M1 to M3 generations is combined with resequencing screening, and stable mutant traits are quickly homozygous within 3 generations, shortening the traditional mapping population construction cycle (usually 5-6 generations of self-pollination), and the efficiency is improved by more than 40%. 10MeV electron accelerator To ensure accurate dose (error <±5Gy), the initial mutagenesis of 350Gy induced a lethality of 15%-20%, and the secondary 150Gy reduced the chimera ratio from 45% to 12%.
[0048] Figure 3 This is the fiber length mutation mapping population for Zhongmian 113;
[0049] The advantages are as follows: for key traits such as cotton fiber length and strength (for example, the fiber length variation in the embodiment is up to ±35 mm), extreme phenotypic populations are constructed, providing high-resolution point mutation materials for gene mapping.
[0050] Figure 4 This is the carding diagram of part of the material in the group of fiber length mutation mapping for Zhongmian 113.
[0051] The results are as follows: mutants with a fiber length of 34.5 mm were screened out from the M3 generation of some materials (the control cotton 113 was 30.8 mm), and the strength was increased to 35 cN / tex (the original variety was 31 cN / tex), reaching the level of Australian cotton.
[0052] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.
Claims
1. A method for constructing a point mutation mapping population using the embryo cold shock double luring method, characterized in that: The following steps are involved: (1) Select conventional cotton varieties that are currently widely used in agricultural production and have been accurately sequenced; (2) selecting high-quality dry seeds from the cotton varieties of step (1), subjecting them to mutagenesis treatment, and obtaining primary mutagenized M0 generation seeds; (3) The primary mutagenized M0 generation seeds were soaked in gibberellin GA3 at 25°C for 6 h, then fished out and subjected to secondary mutagenization treatment after cold fluorescent irradiation at 18°C for 12 h to obtain secondary mutagenized M0 generation seeds; (4) Sowing the secondary mutagenesis M0 generation seeds, taking young leaves at the three-leaf and one-heart stage, resequencing them against the sequencing reference genome, performing point mutation detection, screening individual plants with target traits, and harvesting mutant M1 generation seeds; (5) From all seeds of the M1 generation harvested from single sowing, young leaves at the three-leaf, one-heart stage were taken and resequenced against the sequencing reference genome for point mutation detection and mutant M2 seeds were harvested from individual plants; (6) From all seeds of the M2 generation harvested from single sowing, young leaves at the three-leaf, one-heart stage were taken and resequenced against the sequencing reference genome for point mutation detection and mutant M3 seeds were harvested from individual plants; (7) All seeds of the M3 generation harvested from single seed sowing were screened for extreme cases of the target trait, and young leaves at the three-leaf, one-heart stage were taken and resequenced against the sequencing reference genome for point mutation detection; (8) Based on the specific situation of point mutation detection, rationally screen the mutation mapping population.
2. The method according to claim 1, characterized in that The cotton variety in step (1) is Zhongmian 113 or Zhongmian 24.
3. The method according to claim 1, characterized in that The mutagenic treatment in step (2) and step (3) is carried out by irradiation with electron beam, laser, X-ray, gamma ray, cosmic ray or high-energy electron linear acceleration, or treatment with EMS, alkylating agent, azide compound, base analogue or nitroso compound.
4. The method according to claim 1, wherein The mutagenesis treatment in step (2) is a high-energy electron linear acceleration irradiation method with an irradiation dose of 350 Gy and an irradiation time of 2 s.
5. The method according to claim 1, wherein The secondary mutagenesis treatment in step (3) is a high-energy electron linear acceleration irradiation method with an irradiation dose of 150 Gy and an irradiation time of 2 s.
6. The method according to claim 1, characterized in that The concentration of gibberellin GA3 in step (3) is 120 ppm.
7. The method according to claim 1, characterized in that The target traits screened in step (4) include leaf shape, size and color, plant height, seed size, cotton fiber length, strength, micronaire value and lint percentage.