Method for high-throughput targeted identification of M1-generation mutation of physicochemical mutated plant and obtaining of mutant

A high-throughput, physical and chemical technology, applied in the field of identification and mutant acquisition, can solve the problem of low mutation efficiency of target genes, and achieve the effect of saving time and cost

Active Publication Date: 2020-03-24
HUNAN HYBRID RICE RES CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Based on the irreplaceable advantages of mutagenesis breeding, we hope to solve the pr

Method used

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  • Method for high-throughput targeted identification of M1-generation mutation of physicochemical mutated plant and obtaining of mutant
  • Method for high-throughput targeted identification of M1-generation mutation of physicochemical mutated plant and obtaining of mutant
  • Method for high-throughput targeted identification of M1-generation mutation of physicochemical mutated plant and obtaining of mutant

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Experimental program
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Effect test

Embodiment 1

[0048] a kind of like figure 1 The high-throughput targeted identification of physical and chemical mutagenesis plant M1 generation mutations of the present invention and the method for obtaining mutants specifically include the following steps:

[0049] 1. Use 80MeV / u carbon ion ( 12 C 6+ ) with a dose of 180Gy to irradiate 20,000 seeds of rice variety 638S to obtain the M1 generation of rice variety 638S (hereinafter referred to as 638S).

[0050] 2. Plant 20,000 seedlings of the 638S M1 generation in individual plants. Take 96 plants as a group, each group has 8 rows, and each row has 12 plants, 100 groups are planted, a total of 9600 plants, and each group is numbered 1-100.

[0051] 3. Taking each group as a unit, take an equal amount of leaves from each individual plant, mix a total of 96 equal parts in a centrifuge tube to extract DNA, and extract a total of 100 copies of DNA, and the DNA number corresponds to the number 1-100 of each group .

[0052] 4. Using the ...

Embodiment 2

[0069] a kind of like figure 1 The high-throughput targeted identification of physical and chemical mutagenesis plant M1 generation mutations of the present invention and the method for obtaining mutants specifically include the following steps:

[0070] 1. Take 20,000 seeds of rice variety Huahang 31, soak the seeds in clear water for 16 hours in a constant temperature incubator at 28°C, remove the seeds and control the moisture. The seeds were again soaked in 1% (w / w) ethyl methanesulfonate (EMS) solution for 8 hours in a constant temperature incubator at 28° C., and the seeds were fished out and dried. Rinse the seeds with clear water, change the water 8 times, and plant after germination at 37°C for sowing and seedling raising. Take 96 plants as a group, each group has 8 rows, and each row has 12 plants. Populations were numbered 1-100.

[0071]2. Taking each group as a unit, take an equal amount of leaves from each individual plant, mix 96 equal parts in a centrifuge tu...

Embodiment 3

[0083] a kind of like figure 1 The high-throughput targeted identification of physical and chemical mutagenesis plant M1 generation mutations of the present invention and the method for obtaining mutants specifically include the following steps:

[0084] 1. use 60 Co-γ rays irradiated 100,000 seeds of rice variety Huazhan with a dose of 350Gy to obtain the M1 generation of rice variety Huazhan.

[0085] 2. After cultivating 100,000 seeds of Huazhan M1 generation, 96 plants are used as a group, each group has 8 rows, each row has 12 plants, and 500 groups are planted, with a total of 48,000 plants, and each group is numbered 1 -500.

[0086] 3. Taking each group as a unit, take an equal amount of leaves from each individual plant, mix a total of 96 equal parts in a centrifuge tube to extract DNA, and extract a total of 500 copies of DNA, and the DNA number corresponds to the number 1-500 of each group .

[0087] 4. Using the DNA of each population as a sequencing sample, co...

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Abstract

The invention discloses a method for high-throughput targeted identification of M1-generation mutation of a physicochemical mutated plant and obtaining of a mutant. The identification method comprisesthe following steps: mutagenizing a plant in a non-lethal dosage physicochemical mutagenesis mode, planting the obtained M1-generation plant into single plants, mixing the leaves of each single plant, extracting polled DNA, and performing high-depth targeted sequencing on a target gene region; and comparing the obtained sequencing result with the related sequence of a target gene region, and identifying whether the target SNP and/or Indel exists or not. DNA identification of the chimeric single plants is carried out on the basis of identifying the chimeric single plants containing target generegion mutation, selecting ears containing the mutation, harvesting seeds of the ears in a mixed mode, performing mixed sowing, and then carrying out DNA identification on the single plants to obtainan M2 single plant with a target genetic phenotype. The method has the advantages of high efficiency, good accuracy, and simplicity in operation, and is of remarkable progressive significance for identification and acquisition of innovative germplasm.

Description

technical field [0001] The invention belongs to the field of identification of plant mutants, and in particular relates to identification of plant mutants induced by physical and chemical mutagenesis and a method for obtaining the mutants. Background technique [0002] Germplasm resources are the basis of plant genetics and breeding. Mutation breeding can produce rich genetic variation, and the resulting variation rate is tens of thousands of times that of natural variation. According to the statistics of the World Atomic Energy Agency in 1985, countries around the world had bred more than 500 varieties through mutagenesis at that time, and obtained a large amount of valuable germplasm resources at the same time. However, there are also many obvious defects in mutation breeding, the most important of which is that the mutation position is random, and the target mutant can only be selected in the M2 generation, which leads to low accuracy and efficiency of innovative germplas...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895A01H1/06
CPCA01H1/06C12Q1/6895C12Q2600/156
Inventor 韶也赵炳然毛毕刚彭彦唐丽李曜魁胡远艺张丹柏连阳袁隆平
Owner HUNAN HYBRID RICE RES CENT
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