CN105918114A - Method for improving screening efficiency of target traits in plant radiation mutation breeding - Google Patents
Method for improving screening efficiency of target traits in plant radiation mutation breeding Download PDFInfo
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- A—HUMAN NECESSITIES
- 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
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Abstract
The invention discloses a method for improving screening efficiency of target traits in plant radiation mutation breeding. The method comprises the following steps: conducting radiation mutation treatment on plant seeds to obtain M0 seeds; subjecting the M0 seeds to multiple-sample coarse transplanting, collecting main spike seeds to obtain M1 seeds, conducting self seed-setting on M1 seeds to obtain M2 seeds; subjecting M2 seeds to seedling culture in indoor, growing the seedling to two leaves with one bud, and extracting genomic DNA from each individual plant. The characteristic of radiation mutation accelerating the mutation rate of an organism's genome is used; in the seedling stage, molecular biology technique is used for directed screening of target trait mutants from radiation mutation group, so as to reduce the work lead for gene without mutation or mutation unqualified for the target traits, and improve the accuracy of screening of target trait mutants.
Description
Technical field
The present invention relates to a kind of foliage filter screening method, be specifically related to a kind of raising plant radiation mutagenic breeding objective trait directed screening
The method of efficiency.
Background technology
Crops radiaction mutation is guaranteeing that world food safety and the supply respect that has additional nutrients have important effect.According to connection
Close state FAO/IAEA mutating variety data base's recent statistics, by the end of in by the end of September, 2013, have more than 60 country at 214
It is bred as on floristics and passes through the plant mutation kind sum of business registration having reached 3218, the most directly or indirectly utilize induction
The new varieties that mutant is cultivated are respectively 274,312,824 in world's chief crop Semen Tritici aestivi, Fructus Hordei Vulgaris and Oryza sativa L., account for whole
Mutation is bred as nearly the 50% of mutating variety number.Under long-term natural environmental condition, organism self and external environment phase interaction
With, in order to adapt to the change of environment, its internal hereditary material can occur certain spontaneous mutation, but frequency is the lowest, is about
10-5～10-8Secondary.And utilize core Mutation induction technology inducing plant genome produce sudden change or cause chromosomal aberration and structure variation,
Improve Mutation induction frequency, it is thus possible to create character, the type made new advances, enrich germplasm resource bank and widen biological heredity multiformity,
Generation nature can also be induced rare or be difficult to the new gene type obtained with conventional breeding methods, the selection-breeding for new varieties provides rich
Rich original material.Plant germplasm resource lacks the bottleneck becoming restriction plant breeding, and radioinduction technology has become creating plants
How the effective way of germ plasm resource, due to the randomness of radioinduction, improve mutagenic frequency, carry out the fast of required objective trait
Speed, efficient screening become the problem that breeder pays close attention to the most.
Summary of the invention
The shortcoming of prior art in view of the above, it is an object of the invention to provide a kind of raising plant radiation mutagenic breeding target
The method of character directed screening efficiency, to overcome the defect that in prior art, breeding speed is slow, breeding efficiency is the highest.
In order to reach foregoing invention purpose and other purposes, the present invention is achieved by the following technical solutions:
A kind of method improving plant radiation mutagenic breeding objective trait directed screening efficiency, comprises the steps:
1) plant seed is carried out radioinduction process and obtains M0 for seed；
2) M0 carries out many most slotting plantation only main fringe seeds of mixed receipts for seed and obtains M1 generation, and M1 obtains M2 generation for self-fertility
Seed；
3) M2 carries out sending out Seedling for seed and cultivates in indoor, until growth of seedling to two leaves wholeheartedly time, extract the base of each individual plant respectively
Because of group DNA；
4) determine Breeding objective, Gene bank data base searches the CDNA sequence of objective trait gene；
5) carried out the design of molecule primers by Primer Premier software according to the CDNA sequence of objective trait gene；
6) with the molecule primers optimized, through PCR process, M2 is expanded for the genomic DNA of individual plant；
7) amplified production carries out agarose gel electrophoresis, compares with DNA MARK, and the molecular size range of detection amplified production is
No in the same size with objective trait gene CDNA acid molecules amount；
8) cut glue recovery and carry out gene order-checking, with objective trait gene CDNA sequence alignment；
9) there is the M2 with objective trait gene CDNA sequence difference for individual plant, be further carried out phenotypic evaluation；
10) strict self propagated offspring, finally selects inheritance stability, and objective trait meets the individual plant of breeding requirement.
Preferably, described plant seed is rice paddy seed.
Preferably, described rice paddy seed carries out immersion treatment to showing money or valuables one carries unintentionally before radioinduction processes, and soak time is for being not less than 24h.
Preferably, described radioinduction source is60Co-gamma-rays.
Preferably, in radioinduction, mutagenic agent dosage is 150～250Gy, and close rate is 1.0～2.0Gy/Min.
Preferably, described objective trait is low cadmium-accumulation.
The present invention utilizes radioinduction can accelerate the feature of organism genome mutation rate, seedling stage use Protocols in Molecular Biology from
Directed screening objective trait mutant in radioinduction colony, decreases consuming and does not meets required target at gene without variation or variation
Character causes the workload in sudden change, and improves the accuracy rate to objective trait screening mutant.
Accompanying drawing explanation
Fig. 1 is mutant HN186-M2-780 and wild type OsNRAMP5 gene CDNA sequence alignment result；
Fig. 2 is mutant HN186-M2-4711 and wild type OsNRAMP5 gene CDNA sequence alignment result；
Fig. 3 is M0 generation60Co-gamma-ray and mutagenesis treatment effect；
Fig. 4 is techniqueflow chart.
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art can be by disclosed by this specification
Content understand other advantages and effect of the present invention easily.The present invention can also be added by the most different detailed description of the invention
To implement or application, the every details in this specification can also be based on different viewpoints and application, in the essence without departing from the present invention
Various modification or change is carried out under god.
Embodiment 1
A kind of method improving plant radiation mutagenic breeding objective trait directed screening efficiency, comprises the steps:
1) plant seed is carried out radioinduction process and obtains M0 for seed；
2) M0 carries out many most slotting plantation only main fringe seeds of mixed receipts for seed and obtains M1 generation, and M1 obtains for strict self-fertility
M2 is for seed；
3) M2 carries out sending out Seedling for seed and cultivates in indoor, until growth of seedling to two leaves wholeheartedly time, extract the base of each individual plant respectively
Because of group DNA；
4) determine Breeding objective, Gene bank data base searches the CDNA sequence of objective trait gene；
5) carried out the design of molecule primers by Primer Premier software according to the CDNA sequence of objective trait gene；
6) with the molecule primers optimized, through PCR process, M2 is expanded for the genomic DNA of individual plant；
7) amplified production carries out agarose gel electrophoresis, compares with DNA MARK, and the molecular size range of detection amplified production is
No in the same size with objective trait gene CDNA acid molecules amount；
8) cut glue recovery and carry out gene order-checking, with objective trait gene CDNA sequence alignment；
9) there is the M2 with objective trait gene CDNA sequence difference for individual plant, be further carried out phenotypic evaluation；
10) strict self propagated offspring, finally selects inheritance stability, and objective trait meets the individual plant of breeding requirement.
Specifically, described plant seed is rice paddy seed.
Specifically, described rice paddy seed carries out immersion treatment to showing money or valuables one carries unintentionally before radioinduction processes, and soak time is 24h.
Specifically, described radioinduction source is60Co-gamma-rays.
Specifically, in radioinduction, mutagenic agent dosage is 250Gy, and close rate is 2.0Gy/Min.
Embodiment 2
A kind of method improving plant radiation mutagenic breeding objective trait directed screening efficiency, comprises the steps:
1) plant seed is carried out radioinduction process and obtains M0 for seed；
2) M0 carries out many most slotting plantation only main fringe seeds of mixed receipts for seed and obtains M1 generation, and M1 obtains for strict self-fertility
M2；
3) M2 carries out sending out Seedling for seed and cultivates in indoor, until growth of seedling to two leaves wholeheartedly time, extract the base of each individual plant respectively
Because of group DNA；
4) determine Breeding objective, Gene bank data base searches the CDNA sequence of objective trait gene；
5) carried out the design of molecule primers by Primer Premier software according to the CDNA sequence of objective trait gene；
6) with the molecule primers optimized, through PCR process, M2 is expanded for the genomic DNA of individual plant；
7) amplified production carries out agarose gel electrophoresis, compares with DNA MARK, and the molecular size range of detection amplified production is
No in the same size with objective trait gene CDNA acid molecules amount；
8) cut glue recovery and carry out gene order-checking, with objective trait gene CDNA sequence alignment；
9) there is the M2 with objective trait gene CDNA sequence difference for individual plant, be further carried out phenotypic evaluation；
10) strict self propagated offspring, finally selects inheritance stability, and objective trait meets the individual plant of breeding requirement.
Specifically, described plant seed is rice paddy seed.
Specifically, described rice paddy seed carries out immersion treatment to showing money or valuables one carries unintentionally before radioinduction processes, and soak time is for being not less than 30h.
Specifically, described radioinduction source is60Co-gamma-rays.
Specifically, in radioinduction, mutagenic agent dosage is 200Gy, and close rate is 1.0Gy/Min.
Embodiment 3
A kind of method improving plant radiation mutagenic breeding objective trait directed screening efficiency, comprises the steps:
1) plant seed is carried out radioinduction process and obtains M0 for seed；
2) M0 carries out many most slotting plantation only main fringe seeds of mixed receipts for seed and obtains M1 generation, and M1 obtains for strict self-fertility
M2；
3) M2 carries out sending out Seedling for seed and cultivates in indoor, until growth of seedling to two leaves wholeheartedly time, extract the base of each individual plant respectively
Because of group DNA；
4) determine Breeding objective gene, Gene bank data base searches the CDNA sequence of objective trait gene；
5) carried out the design of molecule primers by Primer Premier software according to the CDNA sequence of objective trait gene；
6) with the molecule primers optimized, through PCR process, M2 is expanded for the genomic DNA of individual plant；
7) amplified production carries out agarose gel electrophoresis, compares with DNA MARK, and the molecular size range of detection amplified production is
No in the same size with objective trait gene CDNA acid molecules amount；
8) cut glue recovery and carry out gene order-checking, with objective trait gene CDNA sequence alignment；
9) there is the M2 with objective trait gene CDNA sequence difference for individual plant, be further carried out phenotypic evaluation；
10) strict self propagated offspring, finally selects inheritance stability, and objective trait meets the individual plant of breeding requirement.
Specifically, described plant seed is rice paddy seed.
Specifically, described rice paddy seed carries out immersion treatment to showing money or valuables one carries unintentionally before radioinduction processes, and soak time is for being not less than 36h.
Specifically, described radioinduction source is60Co-gamma-rays.
Specifically, in radioinduction, mutagenic agent dosage is 175Gy, and close rate is 1.5Gy/Min.
Embodiment 4
The present embodiment is that radioinduction formulates low cadmium-accumulation rice germplasm mutant；
1) full Oryza sativa L. HN186 dry seeds about 3000 is disinfected, and soaks 24 hours with pure water, makes rice paddy seed
Fully water suction, accelerating germination, when seed shows money or valuables one carries unintentionally, use60Co-gamma-ray irradiation mutagenic treatment, dosage 250Gy, close rate is
1.5Gy/Min, it is thus achieved that M0 is for the mutation colony of seed.Take about 50 radiation treatment seeds at random and untreated seed is (right
According to) do a Seedling test at laboratory germination frame, to observe Seedling damage effect to judge mutagenic treatment effect.
2) M0 is for planting seed seedling, until SANYE wholeheartedly time, slightly insert that plantation is only mixed receives main fringe seeds and obtain M1 generation, M1 for many
For strict self-fertility acquisition M2 for colony's seed, take M2 and carry out sending out Seedling cultivation in indoor for about 5000, colony's seed, treat
Growth of seedling to two leaves wholeheartedly time, extract each mutagenized populations M2 genomic DNA for single-strain blade respectively.
3) search and Cd uptake related gene natural resistance associated macrophages albumen OsNRAMP5 in Gene bank data base
CDNA sequence (ID:AB690551), soft by Primer Premier according to the CDNA sequence of OsNRAMP5 gene
Part carries out the design of specific molecular primer.
4) take mutagenized populations M2 for single-strain blade genomic DNA, be 50ng/ μ l by the concentration dilution of each sample, every 10
Individual sample DNA mixed in equal amounts builds sample cell, and 5000 sample DNAs build 500 mixing pits, number consecutively
500 genomic DNAs, with the specific molecular primer optimized, through PCR process, are mixed by POOL1-POOL500 respectively
Close pond to expand, with the genomic DNA in each sample mix pond as template, carry out PCR according to following system and program,
Obtain amplified production.
The reaction system of amplification and PCR amplification program
Forward primer: 5 '-AGAGAGCAGTGAGAGAGG-3 '；
Reverse primer: 5 '-GACGGAGTCCTTCCTGAT-3 '；
5) amplify 500 PCR primer that each mixing pit is corresponding, carry out agarose gel electrophoresis, with DNA MARK
Relatively, the molecular size range of detection amplified production is the most in the same size with gene CDNA acid molecules amount.
6) cut glue recovery and carry out gene order-checking.
7) use sequence alignment program Bioedit, with wild type OsNRAMP5 gene CDNA sequence alignment, find mixing
Pond POOL78, the sequence of the PCR primer that POOL356, POOL472 are corresponding and wild type OsNRAMP5 gene CDNA
Sequence there are differences.
8) utilize step 4) in forward and reverse primer respectively to mixing pit POOL78, POOL356, POOL472 comprise
In mutagenized populations, 30 M2 carry out PCR amplification for individual plant genomic DNA, and PCR primer carries out agarose gel electrophoresis, with
DNAMARK compares, the molecular size range of detection amplified production, cuts glue recovery and carries out gene order-checking, and M2 is for mutagenized populations
Middle numbering HN186-M2-771, HN186-M2-775, HN186-M2-780, HN186-M2-3554, HN186-M2-3558,
It is poor that 7 individual plants of HN186-M2-4711, HN186-M2-4718 and wild type OsNRAMP5 gene CDNA sequence exist
Different.
9) by step 8) in 7 M2 there are differences of genome sequence for individual plant SANYE wholeheartedly time transplant to cadmium content background
It is worth consistent land for growing field crops, strict self propagated offspring, gathers in the crops seed at period of maturation individual plant, carry out the mensuration of cadmium content in seed,
In HN186-M2-780, HN186-M2-4711 seed, cadmium content versus wild type HN186 seed cadmium content reduces,
In HN186-M2-780, HN186-M2-4711 continuation plantation M3 generation, carries out comprehensive identification, finally picks out and selects inheritance stability, comprehensively
The low cadmium-accumulation mutant that character is excellent.
Cadmium in Soil content background value is 1.225mg/Kg, experimental group HN186, HN186-M2-780 and HN186-M2-4711
Content see table shown in:
| Title | Polished rice cadmium content |
| HN186 | 0.523mg.Cd.Kg-1 |
| HN186-M2-780 | 0.469mg.Cd.Kg-1 |
| HN186-M2-4711 | 0.375mg.Cd.Kg-1 |
The principle of above-described embodiment only illustrative present invention and effect thereof, not for limiting the present invention.Any it is familiar with this skill
Above-described embodiment all can be modified under the spirit and the scope of the present invention or change by the personage of art.Therefore, such as
All that in art, tool usually intellectual is completed under without departing from disclosed spirit and technological thought etc.
Effect is modified or changes, and must be contained by the claim of the present invention.
Claims (6)
1. the method improving plant radiation mutagenic breeding objective trait directed screening efficiency, comprises the steps:
1) plant seed is carried out radioinduction process and obtains M0 for seed；
2) M0 carries out many most slotting plantation only main fringe seeds of mixed receipts for seed and obtains M1 generation, and M1 obtains M2 for self-fertility
For seed；
3) M2 carries out sending out Seedling for seed and cultivates in indoor, until growth of seedling to two leaves wholeheartedly time, extract each individual plant respectively
Genomic DNA；
4) determine Breeding objective, Gene bank data base searches the CDNA sequence of objective trait gene；
5) carried out the design forming of molecule primers by Primer Premier software according to the CDNA sequence of objective trait gene
The molecule primers optimized；
6) with the molecule primers optimized, through PCR process, M2 is expanded for the genomic DNA of individual plant；
7) amplified production carries out agarose gel electrophoresis, compares with DNAMARK, the molecular size range of detection amplified production
The most in the same size with objective trait gene CDNA acid molecules amount；
8) cut glue recovery and carry out gene order-checking, with objective trait gene CDNA sequence alignment；
9) there is the M2 with objective trait gene CDNA sequence difference for individual plant, be further carried out phenotypic evaluation；
10) self propagated offspring, finally selects inheritance stability, and objective trait meets the individual plant of breeding requirement.
2. method as claimed in claim 1, it is characterised in that: described plant seed is rice paddy seed.
3. method as claimed in claim 2, it is characterised in that: described rice paddy seed carries out immersion treatment extremely before radioinduction processes
Showing money or valuables one carries unintentionally, soak time is for being not less than 24h.
4. method as claimed in claim 1, it is characterised in that: described radioinduction source is60Co-gamma-rays.
5. method as claimed in claim 1, it is characterised in that: in radioinduction, mutagenic agent dosage is 150～200Gy, and close rate is
1.0～2.0Gy/Min.
6. method as claimed in claim 1, it is characterised in that: described objective trait is low cadmium-accumulation.
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Cited By (8)
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| CN108849479A (en) * | 2018-07-18 | 2018-11-23 | 唐山市农业科学研究院 | A kind of selection of precocity muskmelon seeds |
| CN110476817A (en) * | 2019-09-29 | 2019-11-22 | 上海市农业科学院 | A kind of iris radioinduction dose screening method |
| CN110537488A (en) * | 2019-07-16 | 2019-12-06 | 江苏新梦想生态环境建设股份有限公司 | Detection method for breeding of cinnamomum longepaniculatum improved variety |
| CN110904258A (en) * | 2019-12-03 | 2020-03-24 | 湖南杂交水稻研究中心 | Method for high-throughput targeted identification of physical and chemical mutation plant M1 generation mutation and acquisition of mutant |
| CN111763755A (en) * | 2019-12-16 | 2020-10-13 | 湖南杂交水稻研究中心 | SNP molecular marker of rice cadmium absorption related gene OsNRAMP5 and application thereof |
| CN113584205A (en) * | 2021-07-27 | 2021-11-02 | 哈尔滨工业大学 | Method for rapidly breeding space mutation short-stalk and low-cadmium accumulation rice |
| CN114014919A (en) * | 2021-11-25 | 2022-02-08 | 湖南省核农学与航天育种研究所 | OsNramp5 mutant and screening method and application thereof |
| CN114014920A (en) * | 2021-11-25 | 2022-02-08 | 湖南省核农学与航天育种研究所 | OsRR22 mutant and screening method and application thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108849479A (en) * | 2018-07-18 | 2018-11-23 | 唐山市农业科学研究院 | A kind of selection of precocity muskmelon seeds |
| CN110537488A (en) * | 2019-07-16 | 2019-12-06 | 江苏新梦想生态环境建设股份有限公司 | Detection method for breeding of cinnamomum longepaniculatum improved variety |
| CN110476817A (en) * | 2019-09-29 | 2019-11-22 | 上海市农业科学院 | A kind of iris radioinduction dose screening method |
| CN110904258A (en) * | 2019-12-03 | 2020-03-24 | 湖南杂交水稻研究中心 | Method for high-throughput targeted identification of physical and chemical mutation plant M1 generation mutation and acquisition of mutant |
| CN110904258B (en) * | 2019-12-03 | 2021-08-17 | 湖南杂交水稻研究中心 | Method for high-throughput targeted identification of physical and chemical mutation plant M1 generation mutation and acquisition of mutant |
| CN111763755A (en) * | 2019-12-16 | 2020-10-13 | 湖南杂交水稻研究中心 | SNP molecular marker of rice cadmium absorption related gene OsNRAMP5 and application thereof |
| CN111763755B (en) * | 2019-12-16 | 2021-05-18 | 湖南杂交水稻研究中心 | SNP molecular marker of rice cadmium absorption related gene OsNRAMP5 and application thereof |
| CN113584205A (en) * | 2021-07-27 | 2021-11-02 | 哈尔滨工业大学 | Method for rapidly breeding space mutation short-stalk and low-cadmium accumulation rice |
| CN114014919A (en) * | 2021-11-25 | 2022-02-08 | 湖南省核农学与航天育种研究所 | OsNramp5 mutant and screening method and application thereof |
| CN114014920A (en) * | 2021-11-25 | 2022-02-08 | 湖南省核农学与航天育种研究所 | OsRR22 mutant and screening method and application thereof |
| CN114014920B (en) * | 2021-11-25 | 2022-07-22 | 湖南省核农学与航天育种研究所 | OsRR22 mutant and screening method and application thereof |
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