CN113317198A - Method for constructing hemp mutant library by mutagenesis and molecular marker technology - Google Patents
Method for constructing hemp mutant library by mutagenesis and molecular marker technology Download PDFInfo
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- 235000012766 Cannabis sativa ssp. sativa var. sativa Nutrition 0.000 title claims abstract description 35
- 235000012765 Cannabis sativa ssp. sativa var. spontanea Nutrition 0.000 title claims abstract description 35
- 235000009120 camo Nutrition 0.000 title claims abstract description 35
- 235000005607 chanvre indien Nutrition 0.000 title claims abstract description 35
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- 238000000034 method Methods 0.000 title claims abstract description 32
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- 239000003147 molecular marker Substances 0.000 title claims abstract description 20
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- 238000002703 mutagenesis Methods 0.000 title claims abstract description 18
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- UCONUSSAWGCZMV-UHFFFAOYSA-N Tetrahydro-cannabinol-carbonsaeure Natural products O1C(C)(C)C2CCC(C)=CC2C2=C1C=C(CCCCC)C(C(O)=O)=C2O UCONUSSAWGCZMV-UHFFFAOYSA-N 0.000 claims description 21
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- 229960004242 dronabinol Drugs 0.000 description 16
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- QHMBSVQNZZTUGM-ZWKOTPCHSA-N cannabidiol Chemical compound OC1=CC(CCCCC)=CC(O)=C1[C@H]1[C@H](C(C)=C)CCC(C)=C1 QHMBSVQNZZTUGM-ZWKOTPCHSA-N 0.000 description 3
- 229950011318 cannabidiol Drugs 0.000 description 3
- WVOLTBSCXRRQFR-DLBZAZTESA-N cannabidiolic acid Chemical compound OC1=C(C(O)=O)C(CCCCC)=CC(O)=C1[C@H]1[C@H](C(C)=C)CCC(C)=C1 WVOLTBSCXRRQFR-DLBZAZTESA-N 0.000 description 3
- QXACEHWTBCFNSA-SFQUDFHCSA-N cannabigerol Chemical compound CCCCCC1=CC(O)=C(C\C=C(/C)CCC=C(C)C)C(O)=C1 QXACEHWTBCFNSA-SFQUDFHCSA-N 0.000 description 3
- PCXRACLQFPRCBB-ZWKOTPCHSA-N dihydrocannabidiol Natural products OC1=CC(CCCCC)=CC(O)=C1[C@H]1[C@H](C(C)C)CCC(C)=C1 PCXRACLQFPRCBB-ZWKOTPCHSA-N 0.000 description 3
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- UCONUSSAWGCZMV-HZPDHXFCSA-N Delta(9)-tetrahydrocannabinolic acid Chemical compound C([C@H]1C(C)(C)O2)CC(C)=C[C@H]1C1=C2C=C(CCCCC)C(C(O)=O)=C1O UCONUSSAWGCZMV-HZPDHXFCSA-N 0.000 description 1
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
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- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
- A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
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Abstract
A method for constructing a hemp mutant library by mutagenesis and molecular marker technology belongs to the technical field of seed-use hemp breeding. The method utilizes cobalt (Co) -60 gamma rays, selects seeds with plump seeds, and sets different irradiation doses for treatment. The method comprises the steps of planting and screening under the natural condition of an isolation device and in a greenhouse, harvesting seeds of M1 generations after maturation, selecting offspring according to germination rate and mutation phenotype, and performing auxiliary selection on M1 and M2 generations of materials by combining a molecular marking method. The establishment of the method is beneficial to obtaining the variant strain with the cannabinol content meeting the standard in a short period, improves the construction efficiency of the mutant library, effectively accelerates the breeding process of industrial hemp, and has important significance for developing the gene function research by constructing the industrial hemp mutant library. The method has the advantages of high efficiency, high mutation rate, short construction period and the like, and can be effectively applied to the series researches of industrial hemp mutation group construction, molecular biology, breeding progeny screening and identification and the like.
Description
Technical Field
The invention belongs to the technical field of hemp breeding, and particularly relates to a method for constructing a hemp mutant library by mutagenesis and molecular marker technology.
Background
Industrial hemp is also called hemp in China, and the european union refers to a hemp variety with no drug utilization value and with a tetrahydrocannabinol content of less than 0.3% as industrial hemp. Industrial hemp is a commercial crop with extremely high added value, and has been used in textile, paper making, medicine and other fields for centuries. Particularly, with the adjustment and upgrading of the industrial structure in recent years, the development and application of the raw materials such as fiber, stalk chips and the like in the aspects of buildings, new materials, active ingredients in medical health care cosmetics, seeds in special food health care products and the like are more prominent.
At present, more than 50 varieties of industrial hemp are produced in China, and more than 90 percent of the varieties adopt a conventional hybridization method. In various breeding methods, the hybridization breeding is to screen progeny materials by configuring hybridization combinations and selecting target characters according to the gene recombination principle, but has the advantages of high requirements on the parent range, long breeding period, low efficiency, large workload and wide separation after hybridization. The mutation breeding is mainly to obtain target variant materials according to the gene mutation principle, although the target variant materials have non-tropism compared with the cross breeding, the mutation rate is high, higher variation probability can be obtained in a shorter time, and the breeding process can be effectively shortened. At present, mutation breeding comprises physical mutation and chemical mutation, has a long history of being applied to crop breeding, and with the development of molecular biology technology, the mutation technology is applied to breeding of new crop varieties by combining with molecular marker technology, and the existence or variation rate of a target gene can be detected by detecting a molecular marker, so that the purpose of selecting target characters or detecting variation is achieved, and the method has the advantages of rapidness, accuracy and no interference of environmental conditions. The current industrial hemp breeding research needs to be continuously innovated from both variety character selection and breeding technology, and simultaneously needs to develop functional genomics and other researches around key characters urgently, but the establishment research of domestic industrial hemp mutant libraries is not developed basically, and the establishment of mutant libraries is the basis of functional genomics, and the gene function can be further identified through mutant analysis.
Disclosure of Invention
The invention aims to solve the blank problem of the hemp mutant library in domestic industry and provides a method for constructing the hemp mutant library by mutagenesis and molecular marking technology, which utilizes cobalt (Co) -60 gamma rays to select full seeds and sets different irradiation doses for treatment. The method comprises the steps of planting and screening under the natural condition of an isolation device and in a greenhouse, harvesting M1 generation seeds after maturation, selecting offspring according to germination rate, seedling rate and mutation phenotype, and performing auxiliary selection on M1 and M2 generation materials by combining a molecular marker method.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for constructing a hemp mutant library by mutagenesis and molecular marker technology comprises the following steps:
the method comprises the following steps: seed selection: selecting more than or equal to 1000 industrial hemp seeds which are uniform in size, complete and full, free from invasion of diseases and pests and mature in development;
step two: and (3) mutagenesis treatment: treating industrial hemp seeds by using Co-60 gamma rays, wherein the treatment dose is 50-200 Gy, the dose rate is 0.1Gy/min, a control group is arranged, and germination and grouped sowing tests are carried out on the mutagenized seeds within 3 days after treatment according to the treatment concentration;
step three: planting and screening treatment: planting in a bag or a greenhouse in a field under natural conditions in an isolated manner by using a seed cultivation frame until M1 generation seeds are harvested;
step four, screening mutant materials:
(1) primarily screening a proper mutagenesis treatment dosage according to the germination condition and the emergence condition of the germination day 7;
(2) counting the mutation phenotype condition by taking 0Gy as a reference according to the material mutation condition from the beginning of the seedling stage to the mature stage of the seeds;
(3) taking the topmost leaves or inflorescences of the plants from the seedling stage to the full-bloom stage, performing qualitative and semi-quantitative detection on the THC and THCA content of the single plants by using a rapid detection technology, removing single plant materials with THC + THCA more than or equal to 0.3%, and further screening the residual materials;
(4) in the full-bloom stage, the top flower and leaf parts of the plants are further subjected to qualitative and quantitative analysis on the contents of THC, THCA, CBD, CBDA, CBG and CBN by using a high performance liquid chromatography technology, wherein THC + THCA is less than or equal to 0.3 percent as a standard for screening, materials meeting the standard are reserved, and materials not meeting the standard are removed;
step five, identifying mutant materials:
(1) collecting seeds of single plants obtained by M1 generation under different treatment doses, taking radicles in a bud stage or leaf pieces in a seedling stage after planting, and quickly freezing by using liquid nitrogen to obtain RAPD molecular marker sample materials;
(2) performing molecular identification on the screened M1 generation plants, and obtaining and constructing M1 generation mutant groups from materials according to the molecular identification result of M1 generation and the mutation phenotype mutation condition;
(3) harvesting seeds of the M1 generation mutant group single plants, planting the seeds into plant rows respectively, and constructing M2 generation groups, wherein the planted M1 and M2 generation groups are subjected to THC + THCA content material drawing and detection, and the material drawing and detection are carried out for 3 and 4 in the fourth step;
(4) after THC and THCA content of M2 generation population is measured in seedling stage, randomly selecting single plant leaves according to plant rows to carry out RAPD molecular marker material taking and identification, wherein the single plant sampling amount is not less than 10% of the population quantity;
(5) and (3) carrying out agronomic trait phenotype identification analysis on the M2 generation material, and screening and constructing an M2 generation mutant library by combining a molecular identification result.
Further, in step four (1), the preliminary screening criteria are: the germination rate is more than or equal to 50 percent, the dead seedling rate is less than or equal to 30 percent, and the seedling protection rate is more than or equal to 70 percent.
Further, in the step four (2), the seedling stage refers to a true leaf stage of 3-5 pairs.
Further, in the step five (2), firstly, screening and obtaining a material with a mutant phenotype from M1 generations primarily according to the change of the mutant phenotype, simultaneously carrying out THC + THCA content measurement, obtaining an M1 generation mutant material with a phenotype and content both meeting the standard from the material, and finally carrying out molecular level detection on all materials of M1 generations with phenotypes and contents both meeting the standard by using a molecular marking method to obtain a M1 generation real mutant material.
Compared with the prior art, the invention has the beneficial effects that: the establishment of the method is beneficial to obtaining the variant strain with the cannabinol content meeting the standard in a short period, the establishment efficiency of the mutant library is improved, the breeding process of the industrial hemp is effectively accelerated, and the establishment of the industrial hemp mutant library has important significance for developing the gene function research. The method has the advantages of high efficiency, high mutation rate, short construction period and the like, and can be effectively applied to the series researches of industrial hemp mutation group construction, molecular biology, breeding progeny screening and identification and the like.
Detailed Description
The technical solutions of the present invention are further described below with reference to the following examples, but the present invention is not limited thereto, and any modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Example 1:
a method for constructing a hemp mutant library by mutagenesis and molecular marker technology comprises the following steps:
the method comprises the following steps: seed selection: selecting more than or equal to 1000 industrial hemp seeds which are uniform in size, complete and full, free from invasion of diseases and pests and mature in development; the number of the selected hemp seeds is enough, so that enough mutants can be selected;
step two: and (3) mutagenesis treatment: treating industrial hemp seeds by using Co-60 gamma rays, setting 5 doses of 50, 100, 150, 200 and 250Gy according to germination, variation and lethal conditions, setting the dose rate to be 0.1Gy/min, and setting a control group, wherein the mutagenized seeds are subjected to germination and grouping sowing tests within 3 days after treatment according to treatment concentration, so that the mutagenic effect can be prevented from declining; of the above treatment doses, 250Gy is the total lethal dose, which does not germinate and emerge, and the other doses are set for comparison and uniform spotting;
step three: planting and screening treatment: planting in a bag or a greenhouse in a field under natural conditions in an isolated manner by using a seed cultivation frame until M1 generation seeds are harvested;
step four, screening mutant materials:
1. primarily screening the appropriate mutagenesis treatment dosage according to the germination rate (7 th day of germination) of more than or equal to 50% and the emergence condition (the death rate of less than or equal to 30% and the seedling protection rate of more than or equal to 70%);
2. counting the mutation phenotype condition (the specific content is shown in table one) by taking 0Gy as a reference from the beginning of a seedling stage (3-5 to true leaves) to the mature stage of seeds according to the material mutation condition;
3. taking the topmost leaves or inflorescences of the plants from the seedling stage to the full-bloom stage, performing qualitative and semi-quantitative detection on the single plants with the THC (tetrahydrocannabinol) and THCA (tetrahydrocannabinolic acid) contents by using a rapid detection technology, removing single plant materials with the THC + THCA being more than or equal to 0.3%, and further screening the residual materials;
4. in the full-bloom stage, the top flower and leaf parts of the plants are further subjected to qualitative and quantitative analysis on the contents of THC, THCA, CBD (cannabidiol), CBDA (cannabidiolic acid), CBG (cannabigerol) and CBN (cannabinol) by using a high performance liquid chromatography technology, wherein the content of THC (%) + THCA (%) is less than or equal to 0.3% for screening, the materials meeting the standard are reserved, and the materials not meeting the standard are removed;
step five, identifying mutant materials:
1. collecting seeds of single plants obtained by M1 generation under different treatment doses, taking radicles in a bud stage or leaf pieces in a seedling stage after planting, and quickly freezing by using liquid nitrogen to obtain RAPD molecular marker sample materials;
2. performing molecular identification on the screened M1 generation plants, and obtaining and constructing M1 generation mutation populations (primers are shown in Table II) from materials according to the molecular identification result of M1 generation and the mutation phenotype mutation condition; firstly, screening a material with a variant phenotype from M1 generation primarily according to the variation of the phenotype after mutagenesis, specifically referring to the table I, and simultaneously carrying out THC + THCA content measurement to obtain a M1 generation variant material with the phenotype + content meeting the standard. Finally, carrying out molecular level detection on all M1 generation materials with phenotype + content meeting the standard by using a molecular marker method, and finally obtaining M1 generation real variant materials;
3. harvesting seeds of the M1 generation mutant group single plants, planting the seeds into plant rows respectively, and constructing M2 generation groups, wherein the planted M1 and M2 generation groups are subjected to THC + THCA content material drawing and detection, and the material drawing and detection are carried out for 3 and 4 in the fourth step;
4. after THC + THCA content of M2 generation population is measured in seedling stage, randomly selecting single plant leaves according to plant rows to carry out RAPD molecular marker material taking and identification (the primer is shown in the same table II), wherein the single plant sample amount is not less than 10% of the population quantity;
5. and (3) carrying out agronomic trait phenotype identification analysis on the M2 generation material, and screening and constructing an M2 generation mutant library by combining a molecular identification result.
Claims (4)
1. A method for constructing a hemp mutant library by mutagenesis and molecular marker technology is characterized in that: the method specifically comprises the following steps:
the method comprises the following steps: seed selection: selecting more than or equal to 1000 industrial hemp seeds which are uniform in size, complete and full, free from invasion of diseases and pests and mature in development;
step two: and (3) mutagenesis treatment: treating industrial hemp seeds by using Co-60 gamma rays, wherein the treatment dose is 50-200 Gy, the dose rate is 0.1Gy/min, a control group is arranged, and germination and grouped sowing tests are carried out on the mutagenized seeds within 3 days after treatment according to the treatment concentration;
step three: planting and screening treatment: planting in a bag or a greenhouse in a field under natural conditions in an isolated manner by using a seed cultivation frame until M1 generation seeds are harvested;
step four, screening mutant materials:
(1) primarily screening a proper mutagenesis treatment dosage according to the germination condition and the emergence condition of the germination day 7;
(2) counting the mutation phenotype condition by taking 0Gy as a reference according to the material mutation condition from the beginning of the seedling stage to the mature stage of the seeds;
(3) taking the topmost leaves or inflorescences of the plants from the seedling stage to the full-bloom stage, performing qualitative and semi-quantitative detection on the THC and THCA content of the single plants by using a rapid detection technology, removing single plant materials with THC + THCA more than or equal to 0.3%, and further screening the residual materials;
(4) in the full-bloom stage, the top flower and leaf parts of the plants are further subjected to qualitative and quantitative analysis on the contents of THC, THCA, CBD, CBDA, CBG and CBN by using a high performance liquid chromatography technology, wherein THC + THCA is less than or equal to 0.3 percent as a standard for screening, materials meeting the standard are reserved, and materials not meeting the standard are removed;
step five, identifying mutant materials:
(1) collecting seeds of single plants obtained by M1 generation under different treatment doses, taking radicles in a bud stage or leaf pieces in a seedling stage after planting, and quickly freezing by using liquid nitrogen to obtain RAPD molecular marker sample materials;
(2) performing molecular identification on the screened M1 generation plants, and obtaining and constructing M1 generation mutant groups from materials according to the molecular identification result of M1 generation and the mutation phenotype mutation condition;
(3) harvesting seeds of the M1 generation mutant group single plants, planting the seeds into plant rows respectively, and constructing M2 generation groups, wherein the planted M1 and M2 generation groups are subjected to THC + THCA content material drawing and detection, and the material drawing and detection are carried out for 3 and 4 in the fourth step;
(4) after THC and THCA content of M2 generation population is measured in seedling stage, randomly selecting single plant leaves according to plant rows to carry out RAPD molecular marker material taking and identification, wherein the single plant sampling amount is not less than 10% of the population quantity;
(5) and (3) carrying out agronomic trait phenotype identification analysis on the M2 generation material, and screening and constructing an M2 generation mutant library by combining a molecular identification result.
2. The method for constructing a library of hemp mutants by mutagenesis and molecular marker technology according to claim 1, wherein: in the step four (1), the preliminary screening standard is as follows: the germination rate is more than or equal to 50 percent, the dead seedling rate is less than or equal to 30 percent, and the seedling protection rate is more than or equal to 70 percent.
3. The method for constructing a library of hemp mutants by mutagenesis and molecular marker technology according to claim 1, wherein: in the step four (2), the seedling stage refers to a true leaf stage of 3-5 pairs.
4. The method for constructing a library of hemp mutants by mutagenesis and molecular marker technology according to claim 1, wherein: in the step five (2), firstly, screening a material with a mutant phenotype from the M1 generation preliminarily according to the change of the mutant phenotype, simultaneously carrying out THC + THCA content measurement to obtain a mutant material with an M1 generation, and finally carrying out molecular level detection on all materials with the phenotype and the content meeting the standard in the M1 generation by using a molecular marking method to obtain a real mutant material with an M1 generation.
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