CN108901835A - A kind of foundation of broom corn millet EMS mutant library and screening assays - Google Patents
A kind of foundation of broom corn millet EMS mutant library and screening assays Download PDFInfo
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- CN108901835A CN108901835A CN201810644546.9A CN201810644546A CN108901835A CN 108901835 A CN108901835 A CN 108901835A CN 201810644546 A CN201810644546 A CN 201810644546A CN 108901835 A CN108901835 A CN 108901835A
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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
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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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
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
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Abstract
The invention discloses a kind of foundation of broom corn millet EMS mutant library and screening assays, belong to agricultural breeding field, by handling EMS the screening of the bright red rotten seed optium concentration of Yi Xuan, build the technology platform of broom corn millet EMS mutagenesis.From 2333 M obtained2For discovery discovery mutating strain series 103 after strain plantation, including leaf color, blade profile, plant height, fertility, fringe type, fringe color, seed coat color, the frequency of mutation 4.4%.Phenotype mutant library abundant is established, mutant resource abundant and novel germplasm can be provided for the research of broom corn millet functional genomics and breeding.
Description
Technical field
The invention belongs to agricultural breeding field, be related to the building of broom corn millet mutant library, more particularly to one kind for broom corn millet into
Row EMS induces the method for establishing mutant library.
Background technique
Broom corn millet (Panicum miliaceum L.) belongs to annual gramineae Panicum, and chromosome number is the different of 2n=4x=36
Source tetraploid has the characteristics that the maturity period is short, drought-enduring, impoverishment tolerant, saline-alkali tolerant, is northern China arid, the semiarid zone people
The characteristic rain fed crops of Main Cultivation and ideal multiple crops.It is known as glutinous millet after broom corn millet shelling, rich in the more of needed by human body
Kind protein, starch, fat etc., nutrition and health care value is high, numerous food can be processed to, for preventing and treating allergy, obesity etc.
Related disease；In addition, its stalk and seed are high-quality domestic animal forage grass and feed again, therefore, meeting popular food consumption diversification
In demand and animal husbandry development, broom corn millet is occupied an important position.
Mutation is the important impetus of biological evolution.Mutant can also provide material for the research of gene function.With
Many methods for analyzing and identifying gene function have been developed in the research and development of constantly bringing forth new ideas of new technology, wherein most effective most direct
Method is the gene mutation body library of building saturation.Building saturated mutant library is to carry out Functional Plant Genomics and genetic breeding
One of the direct and effective method of research.Chemical mutagen ethylmethane sulfonate (EMS) is a kind of stabilization, and efficient chemistry lures
Become agent, the range that EMS is used is wide, it can handle the different tissues and organ such as seed, seedling, pollen or callus.EMS
The mutation rate that mutagenesis generates is high, is not necessarily to genetic transformation, point mutation easy to form, and mostly dominant mutation, is easy to the sieve of mutant
Choosing, does not easily cause chromosome aberration.Currently, EMS chemical mutagenesis method has been applied successfully to arabidopsis, rice, wheat, jade
In the plants such as rice, sorghum, obtain the valuable innovating germplasm resource of a batch, improvement plant growth period, it is disease-resistant with resistance,
Major progress is achieved in terms of the characters such as setting percentage, plant height.The screening and identification of these mutant is that crop gene group will be ground from now on
Study carefully and provide material foundation abundant with crop genetic improvement, there is important scientific practice meaning.Construct broom corn millet EMS mutant
After library, the breeding material for the kind genetic improvement can be obtained by the identification of biological character and economical character, screening
Material, meanwhile, research material abundant is provided for forward genetics and the functional genomics analysis of broom corn millet.
Broom corn millet is better than other C4 plants such as millet, sorghum to the utilization rate of moisture and its Characteristics of Drought, is to carry out gene
The good material of group research.However, domestic start late to broom corn millet Gene mining and Function Identification, has no at present and broom corn millet is mutated
The report of the building in body library, identification and its functional genome.
Summary of the invention
To solve the above-mentioned problems of the prior art, the object of the present invention is to provide a kind of broom corn millet EMS mutant libraries
Foundation and screening assays, establish phenotype broom corn millet mutant library abundant, help speed up and excavate many merits of broom corn millet
Gene process, and mutant material abundant is provided for the research of broom corn millet functional genomics and breeding.
To achieve the above object, the present invention provides following schemes：
The present invention provides foundation and the screening assays of a kind of broom corn millet EMS mutant library, it is characterised in that：According to following
Step carries out：
(1)
The preparation of seed：The bright red rotten seed of full seed, uniform Yi Xuan is chosen, the bright red rotten seed two of Yi Xuan is added
The distilled water of times volume is placed in refrigerator after impregnating 6h~8h at 4 DEG C after shaking up and takes out, is placed at room temperature for 10min；
(2) EMS is induced：Under the conditions of draught cupboard, 1%EMS solution is added in seed, under room temperature in oscillator
300rpm/min mixing oscillation 10h；EMS solution process container is taken out, is added and the isometric 0.2M Na of EMS solution2S2O3In
With, it turns upside down after mixing 7-10 times, standing 15min；The EMS aqueous solution neutralized is filtered out with strainer device, waste liquid pours into logical
In being lauched of wind cupboard, by seed plus distilled water flushing 5 times, water is filtered with strainer device every time it is clean, until the solution of outflow does not have
There is foam-like；Handled seed is poured into strainer, is placed in wash pool from draught cupboard taking-up, rinses 5min with flowing water, it is empty
In hang it is drained；
(3) mutagenesis seed is planted：By through EMS induction, treated that seed is known as M in step (2)1For seed, M is planted1Generation
Seed, line-spacing 30cm, spacing in the rows 6cm；The time of infertility observes and records M1Plant damage and catastrophe, 10 months harvest single plant kinds
Son, these seeds are known as M2Generation；
(4) mutant plant correlated traits is investigated：
The mutant library M that plantation step (3) obtains2Generation, 15-20 plants of every strain final singling are pair with the bright red gruel of wild Yi Xuan
According to material, to M2The growth variability situation in generation carries out careful field observation, the different times plant from after seed sprouting
Morphological feature：Seedling Stage counts the ratio of Albino Seedling and etiolated seedling, and the jointing stage observes the incidence of plant, booting heading stage system
Meter downgrades the ratio and the length in maturity period of seedling, and the mutant special in observation of each period form, and it is special to record mutation in detail
Sign and type count the frequency of mutation, carry out single plant harvest to the strain for having obvious character mutation；
(5)M2For frequency of mutation calculation formula：
M2For the frequency of mutation=M2For mutating strain series/M2For group's strain number × 100%.
Preferably, in step (4), the correlated traits is that chlorophyll content in leaf blades is reduced or lacked, and plant height increases or drops
Low, fringe type, fringe color, seed coat color change, and the maturity period shifts to an earlier date or postpones, Fertility change.
Beneficial effects of the present invention：
The method of the present invention, for material, carries out the optium concentration of the bright red rotten seed of EMS processing Yi Xuan with the bright red gruel of Yi Xuan
System research and screening build the technology platform of broom corn millet EMS mutagenesis for the first time and obtain phenotype broom corn millet mutant library abundant.It is right
2333 M2The observation that generation carries out the time of infertility counts display, and material mutation type is abundant, finds 103 morphological characters mutation altogether
Strain, including leaf color, blade profile, plant height, fringe type, fringe color, seed coat color, fertility.
The method of the present invention can obtain phenotype broom corn millet mutant library abundant in a short time, not only help quickening and excavate gruel
Many merit gene processes in sub- can also provide mutant material abundant for the research of broom corn millet functional genomics and breeding
Material.It downgrades and the acquisition of fringe type mutant is then to solve the problems, such as that lodging and seed holding present in production establish material base as early as possible
Plinth.
Detailed description of the invention
Fig. 1 is the morphological feature figure of Yi Xuan of the present invention bright red rotten etiolated seedling and Albino Seedling；
Fig. 2 is the bright red rotten incidence figure of Yi Xuan of the present invention；
Fig. 3 is the bright red rotten early-maturing mutant figure of Yi Xuan of the present invention；
Fig. 4 is the bright red rotten Dwarf Mutant figure of Yi Xuan of the present invention；
Fig. 5 is the bright red rotten fringe type mutant figure of Yi Xuan of the present invention.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, With reference to embodiment
The present invention is described in further detail.
Embodiment 1
1. material to be tested
The bright red gruel of Yi Xuan
2. experimental design
Test sets 6 concentration gradients, and each concentration sets 3 repetitions, the bright red gruel seed of 300 Yi Xuan of each reprocessing, and 1
A clear water control.
3. test method
(1) the bright red rotten seed of full seed, uniform Yi Xuan is chosen, the bright red rotten seed two volumes of Yi Xuan are added
Distilled water is placed on 4 DEG C of immersion 6h in refrigerator after shaking up；
(2) compound concentration is respectively 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2% EMS solution；
(3) the bright red rotten seed of 300 Yi Xuan is handled respectively with prepared EMS solution, under room temperature on the oscillator
Oscillation 10h is mixed with 300rpm/min；
(4) it is added and the isometric 0.2M Na of EMS solution2S2O3It neutralizes, turns upside down after mixing 7-10 times, stand
15min；
(5) the EMS aqueous solution neutralized is filtered out with strainer device, waste liquid pours into being lauched of draught cupboard, by seed plus steaming
Distilled water is rinsed 5 times, every time filters water strainer device completely, until the solution of outflow does not have foam-like；By handled seed
It is poured into strainer (seed mesh bag), is placed in wash pool from draught cupboard taking-up, rinse 5min with flowing water, hang drain water in the air
Point；
(6) by through EMS induction, treated that seed is known as M in step (5)1For seed, M is planted1For seed, line-spacing 30cm,
Spacing in the rows 6cm, the time of infertility observe and record M1Plant damage, such as Albino Seedling and etiolated seedling, emergence and catastrophe；It harvests within 10 months
Single-strain seed amounts to 2333, these seeds are known as M2Generation.
4. experimental result counts
Different EMS concentration are counted after emergence treated seed number of seedling, and calculate seeding rate, as a result see the table below 1.
The influence of 1 difference EMS concentration of table processing rotten seed planting percent bright red to Yi Xuan
The determination of the most suitable concentration for the treatment of of 5.EMS
It is generally acknowledged that half lethal dose (dosage of plant survival 50% i.e. after EMS mutagenic treatment) is best mutagens
Amount, to be led to the heritable energy threshold that makes a variation and absorb.From table 1 it follows that after 1.0% EMS concentration processing, she
Select the lethality of bright red gruel closest to 50%, accordingly, it is determined that 1.0% EMS concentration is most suitable concentration for the treatment of.
6.M2It is planted for seed
The mutant library M that plantation step (6) obtains2For seed, 15-20 plants of every strain final singling, with the bright red gruel of wild Yi Xuan
For control material, to M2The growth variability situation in generation carries out careful field observation, and different times are planted from after seed sprouting
The morphological feature of strain：
It is 2% that Seedling Stage, which counts Albino Seedling and the ratio of etiolated seedling, and the morphological feature figure of etiolated seedling and Albino Seedling is shown in Fig. 1
It is shown；Jointing stage observes the incidence of plant, and specific form as shown in Figure 2, has in jointing stage plant easily susceptible as the result is shown
The case where with blade tip fracture；Booting heading stage statistics downgrades ratio and the time in maturity period of seedling, wherein M2It is sent out altogether in group
Mutation relevant to plant height has occurred in existing 15 strains, in addition the frequency of mutation 0.6% is also observed precocious and late-maturing each respectively
3 mutating strain series, mutation rate 0.03%.
And the mutant special in Seedling Stage, jointing stage, booting heading stage and maturity period observation form, early-maturing mutant is such as
Shown in Fig. 3, having a strain is Super prematurity mutating strain series, after planting only begins to ear within 24 days or so, compares material than same cell
Material heading period about 30 days in advance.Dwarf Mutant is as shown in figure 4, short bar is mutated：Due to current year, rainfall is plentiful, the bright red gruel of Yi Xuan
Average plant height reaches 167cm, and observation discovery Dwarf Mutant occurs in 8 strains, the highest 141cm of these mutant, most
Short 89cm, only the 1/2 of wild type, fringe type mutant is as shown in figure 5, the bright red rotten fringe type of wild type Yi Xuan is side fringe type, pumping
The whole lateral side of fringe is sagging after fringe.Its average spike length is 33.2cm, and average spike weight is 6.5g.In M since heading stage2Dai Fa
The mutant of 4 seed types is observed in the mutating strain series relevant to fringe type of existing 15 altogether：(1) the close mutant of fringe：Totally 5 mutation
Strain is scattered as wild type to a direction though showing as full fringe, and fringe top set is shorter, and small ear arrangement is compact.Fringe is close
The average spike length of mutant is 30.4cm, and average spike weight is 10.5g.(2) the upright mutant of fringe：Totally 3 mutating strain series, performance
For mutant until maturation since heading, full fringe is not only thin but also long and upright.The average spike length of the upright mutant of fringe is
30.4cm, average spike weight are 1.9g.(3) the thick mutant of fringe：Totally 2 mutating strain series, relative to the bright red gruel of wild type Yi Xuan
Side fringe type, it is extremely short that thick fringe mutant shows as the fringe top set since heading, so that the arrangement of each branch's small ear is very compact, in group
Shape is upward, is similar to sorghum fringe.The average spike length of the thick mutant of fringe is 17.7cm, and average spike weight is 5.1g.(4) fringe radiation is prominent
Variant：Totally 5 mutating strain series all irregularly scatter around from heading to maturity period whole fringe.
The frequency of mutation is counted, single plant harvest is carried out to the strain for having obvious character mutation.
M2It is for frequency of mutation calculation formula：M2For the frequency of mutation=M2For mutating strain series/M2For group's strain number ×
100%.
To 2333 M2The observation that generation carries out the time of infertility counts display, and material mutation type is abundant, finds 103 shapes altogether
State property shape mutating strain series, the frequency of mutation 4.4%, including leaf color, blade profile, plant height, fertility, fringe type, fringe color, seed coat color, tool
Decorum meter such as table 2.
2 M of table2Mutagenesis group phenotypic mutation type statistics
Embodiment described above is only that preferred embodiment of the invention is described, and is not carried out to the scope of the present invention
It limits, without departing from the spirit of the design of the present invention, those of ordinary skill in the art make technical solution of the present invention
Various changes and improvements, should all fall into claims of the present invention determine protection scope in.
Claims (2)
1. foundation and the screening assays of a kind of broom corn millet EMS mutant library, it is characterised in that：It follows the steps below：
(1) preparation of seed：The bright red rotten seed of full seed, uniform Yi Xuan is chosen, the bright red rotten seed two of Yi Xuan is added
The distilled water of times volume is placed in refrigerator after impregnating 6h~8h at 4 DEG C after shaking up and takes out, is placed at room temperature for 10min；
(2) EMS is induced：Under the conditions of draught cupboard, 1%EMS solution is added in seed, under room temperature in oscillator
300rpm/min mixing oscillation 10h；EMS solution process container is taken out, is added and the isometric 0.2M Na of EMS solution2S2O3In
With, it turns upside down after mixing 7-10 times, standing 15min；The EMS aqueous solution neutralized is filtered out with strainer device, waste liquid pours into logical
In being lauched of wind cupboard, by seed plus distilled water flushing 5 times, water is filtered with strainer device every time it is clean, until the solution of outflow does not have
There is foam-like；Handled seed is poured into strainer, is placed in wash pool from draught cupboard taking-up, rinses 5min with flowing water, it is empty
In hang it is drained；
(3) mutagenesis seed is planted：By through EMS induction, treated that seed is known as M in step (2)1For seed, M is planted1For seed,
Line-spacing 30cm, spacing in the rows 6cm；The time of infertility observes and records M1Plant damage and catastrophe, 10 months harvest single-strain seeds, these
Seed is known as M2Generation；
(4) mutant plant correlated traits is investigated：
The mutant library M that plantation step (3) obtains2Generation, 15-20 plants of every strain final singling are bright red rotten for control material with wild Yi Xuan
Material, to M2The growth variability situation in generation carries out careful field observation, the form of different times plant from after seed sprouting
Feature：Seedling Stage counts the ratio of Albino Seedling and etiolated seedling, and the jointing stage observes the incidence of plant, and booting heading stage statistics is short
Change the ratio and the length in maturity period of seedling, and in the special mutant of observation form of each period, record in detail Characteristics of Mutation and
Type counts the frequency of mutation, carries out single plant harvest to the strain for having obvious character mutation；
(5)M2For frequency of mutation calculation formula：
M2For the frequency of mutation=M2For mutating strain series/M2For group's strain number × 100%.
2. requiring foundation and the screening assays of a kind of broom corn millet EMS mutant library according to right 1, it is characterised in that：
In step (4), the correlated traits is that chlorophyll content in leaf blades is reduced or lacked, and plant height increases or reduces, fringe type, fringe color, seed
Grain color change, maturity period shift to an earlier date or postpone, Fertility change.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110100728A (en) * | 2019-05-21 | 2019-08-09 | 中国农业科学院烟草研究所 | A method of mutant library is generated by EMS mutagenesis bluish dogbane |
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| CN107372098A (en) * | 2017-08-21 | 2017-11-24 | 安徽绿亿种业有限公司 | A kind of method of the low low-phosphorous rice varieties of albumen of mutagenic and breeding |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107372098A (en) * | 2017-08-21 | 2017-11-24 | 安徽绿亿种业有限公司 | A kind of method of the low low-phosphorous rice varieties of albumen of mutagenic and breeding |
Non-Patent Citations (2)
| Title |
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| 朱睦元等: "《现代遗传学实验》", 31 May 2009 * |
| 王彩芬等: "水稻种子化学诱变剂EMS浓度筛选研究", 《宁夏农林科技》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110100728A (en) * | 2019-05-21 | 2019-08-09 | 中国农业科学院烟草研究所 | A method of mutant library is generated by EMS mutagenesis bluish dogbane |
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