CN105861734A - Rapid identification method for homozygosis, heterozygosis and resistance-free performance of anti-nicosulfuron genes of foxtail millet - Google Patents
Rapid identification method for homozygosis, heterozygosis and resistance-free performance of anti-nicosulfuron genes of foxtail millet Download PDFInfo
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Abstract
The invention discloses a rapid identification method for the homozygosis, heterozygosis and resistance-free performance of anti-nicosulfuron genes of foxtail millet, and belongs to the technical field of gene engineering. The rapid identification method includes the steps that identification is carried out in the mode that No.1877 deoxyribonucleotide of ALS genes in to-be-detected foxtail millet is detected, genome DNA of the to-be-detected foxtail millet materials is amplified through primer pairs capable of amplifying No.1877 basic groups containing the ALS genes, and PCR amplification products are obtained; the PCR amplification products are subjected to enzyme digestion through restriction enzymes, and enzyme-digested product fragments with different sizes are obtained; the enzyme-digested fragments are viewed through agarose gel electrophoresises, and homozygosis, heterozygosis or sensitive genotypes are identified; if the enzyme-digested fragments are single bands, the to-be-detected foxtail millet materials are non-anti-nicosulfuron foxtail millet materials; if the enzyme-digested fragments are two bands, the to-be-detected foxtail millet materials are homozygosis anti-nicosulfuron foxtail millet materials; if the enzyme-digested fragments are three bands, the to-be-detected foxtail millet materials are heterozygosis anti-nicosulfuron foxtail millet materials. The method is simple, easy to operate, high in identification accuracy and low in cost.
Description
Technical field
The invention belongs to gene engineering technology field, relate to plant gene engineering technology, be specifically related to a kind of Rapid identification
Millet anti-nicosulfuron gene pure, heterozygosis or not anti-method.
Background technology
Millet not antiweed itself, millet planting relies on artificial thinning and weeding always, time-consuming, restriction millet collection
Reduction, large-scale production.The cultivation of herbicide resistance foxtail millet kind, produces significant to millet.In recent years, Hebei province's agriculture
Woods academy of science millet institute has been bred as the Millet Variety of serial anti-imazethapyr herbicide by distant hybridization, it is achieved that
The chemical weed control in paddy field；Meanwhile, proposing, the homotype Sister Lines of sense Sethoxydin anti-by selection-breeding, the two mixes by a certain percentage broadcasts
Kind, utilizing the two Resistant Difference to herbicide, seedling stage synchronizes to achieve millet chemistry thinning by herbicide spraying, chemistry removes
The simplified cultivation millet variety selection of grass and cultivation technique, and be bred as series of products, makes that millet is intensive, scale metaplasia
Product is possibly realized.In producing at present, the herbicide resistance foxtail millet of application is mainly Sethoxydin and imazethapyr type.But make for a long time
There is the potential risk making weeds develop immunity to drugs with single herbicide.Therefore, excavate and utilize and can be used for the new of Millet Breeding
Type herbicide resistance gene, for widening millet antiweed breeding, the multiformity of increase millet Herbicid resistant has important
Meaning.
The anti-cigarette obtained screens in the anti-imazethapyr wild Green foxtail colony that this this problem is introduced from Canada
Sulfometuron Methyl mutant is material, and hybridizes with domestic millet, has formulated anti-nicosulfuron herbicide agent millet new germ plasm YM15.Cigarette is phonetic
Sulphur is grand belongs to sulfonylurea herbicide, is the leading products of ALS (acetolactate synthestase) suppression class herbicide, in world wide
Use, have efficiently, low toxicity, consumption are low, environmentally friendly etc. feature, can effectively prevent and kill off most standing grain annual, perennial this
Section weeds and broad leaved weed and dried tuber.Cultivate anti-nicosulfuron millet and the multiformity adding millet Herbicid resistant is had weight
Want meaning.
But, in order to cultivate the anti-nicosulfuron Millet Variety being capable of millet chemistry thinning and weeding, need choosing
Select anti-, sense nicosulfuron homotype Sister Lines, therefore cannot reach selection-breeding purpose by herbicide spraying.Identify herbicide at present
Anti-, sense, the main method of heterozygous plant have field test method and indoor cultivation ware method.Field test method is i.e. to the part in head progeny row
Plant herbicide spraying, passes through Resistant Difference, it is judged that it is anti-, sense, heterozygosis situation.After indoor cultivation ware method is results seed, take
Part is germinateed for indoor cultivation ware, by herbicide spraying, observes its Resistant Difference, thus judge that it is anti-, sense, heterozygosis feelings
Condition.But these two kinds of method workloads are big, it are unsuitable for high-volume and identify；Meanwhile, the method is easily affected by environmental factors, impact
Judged result.
Summary of the invention
The present invention is directed to deficiency of the prior art, it is provided that a kind of Rapid identification millet anti-nicosulfuron gene pure,
Heterozygosis or not anti-method.
The present invention the technical scheme is that for realizing its purpose
Rapid identification millet anti-nicosulfuron gene pure, heterozygosis, the method for the most anti-type, by detecting millet material to be measured
The base that middle als gene is the 1877th is identified, comprises the following steps:
A, genomic PCR amplification: use amplimer with the genomic DNA of millet material to be measured as template, carry out PCR expansion
Increase, obtain pcr amplification product；
B, enzyme action process: the pcr amplification product obtained with digestion with restriction enzyme, obtain digestion products fragment；
C, the qualification of anti-nicosulfuron gene: check digestion products fragment by agarose gel electrophoresis, if after enzyme action being
Single slice, millet material the most to be measured is the most anti-nicosulfuron millet material；If after enzyme action being two bands, millet material the most to be measured is
Isozygoty anti-nicosulfuron millet material；Being three bands after enzyme action, millet material the most to be measured is heterozygosis anti-nicosulfuron millet material.
The Primer of described amplimer is as follows with nucleotide sequence:
Nicaps-F:3 '-TGCAAGCAGGGCTAAGATTG；
Nicaps-R:3 '-GTACCCCCTTTCATGCACAA.
Described restricted enzyme is PvuI.
The genomic DNA of millet material to be measured is obtained by following method: takes millet blade 100-200mg to be measured, uses CTAB
Method extracts genomic DNA.
In step A genomic PCR amplification, PCR reaction system is: 2 × PCR mix, 10 μ l；Nicaps-F, 0.5 μ l；
Nicaps-R, 0.5 μ l；Millet genomic DNA template to be measured, 1 μ l；ddH2O, 8 μ l；Cumulative volume, 20 μ l.
In step A genomic PCR amplification, PCR response procedures is: 94 DEG C of denaturations 4min, 94 DEG C of degeneration 40s, and 57 DEG C are moved back
Fire 30s, 72 DEG C extend 80s, 35 circulations, 72 DEG C of insulation 7min.
During step B enzyme action processes, enzyme action system is: pcr amplification product, 7 μ l；10 × buffer, 1 μ l；PvuI, 0.2 μ l；
ddH2O, 1.8 μ l；Cumulative volume, 10 μ l, 37 DEG C of enzyme action 30min.
Rapid identification millet anti-nicosulfuron gene pure, heterozygosis or the test kit of the most anti-type, described test kit includes soon
Speed identifies millet anti-nicosulfuron gene pure, heterozygosis or the reagent of non-resistance, and described reagent is by shown in SEQ ID NO.1
The amplified production of DNA molecular composition.
The application in identifying millet nicosulfuron character to be measured of the described test kit.
The invention has the beneficial effects as follows: the present invention can be used for the new herbicides resistance of Millet Breeding by excavation and utilization
Gene, for widen millet antiweed breeding, increase millet Herbicid resistant multiformity significant, the phonetic sulphur of anti-cigarette
The initiative of grand millet kind matter is laid a good foundation for cultivating new herbicides millet.The present invention lives according to anti-nicosulfuron Gene A LS
Property site list base sudden change, establish that qualification accurate, simple and quick is anti-, sense, the method for heterozygosis nicosulfuron millet.With often
Rule indoor cultivation ware is compared with field plant identification method, and it is reliable and stable that the method has result, easy and simple to handle, quick, automatization
The advantage that degree is high.By this molecular mark, anti-nicosulfuron millet can be accelerated and resist, feel the supporting product of homotype Sister Lines
The cultivation planted, shortens breeding cycle, improves breeding efficiency.
Accompanying drawing explanation
Fig. 1 is ALS anti-nicosulfuron active site sequence comparative result figure in difference.
Fig. 2 is pcr amplification product figure.
Fig. 3 is PvuI enzyme action qualification figure.
Fig. 4 is NiALS-CAPs testing result figure in weighing apparatus morning × YM15F2 colony.In accompanying drawing, 1:YM15；2: Heng Gu
No. 12；3-13 is the F2 plant after weighing apparatus morning × YM15 hybridization.
Fig. 5 NiALS-CAPs is marked at the PCR result figure of different cultivars.In accompanying drawing, different cultivars corresponds to respectively: 1:
Ji paddy 19；2: Ji Gu 31；3: Ji Gu 37；4: two-story valley 2；5:S80；6: stone 98622；7: elite No. 5；8: prolong paddy No. 13；9: Henan paddy
18；10: protect 508；11: deep blue 368；12: Jinan Cattle, 13: Jin Gu No. 31；14: Jin Gu No. 32；15: weighing apparatus paddy 12: Datong District 25；
17: Ji 0626: gold Seedling.
Detailed description of the invention
Below in conjunction with Figure of description and specific embodiment, the present invention is further illustrated.With YM15, nicosulfuron
Sensitive material weighing apparatus paddy No. 12 and weighing apparatus paddy No. 12 × YM15F2 offspring be material, establish that a kind of accurate simple and quick qualification is anti-, feel,
The CAPs molecule labelling method of heterozygosis nicosulfuron herbicide agent millet, to accelerating to cultivate anti-nicosulfuron Millet Breeding efficiency, contracting
Short breeding cycle is significant.
The kind of the millet used of the present invention is preserved by Hebei Prov. Academy of Agricultural &. Forest Sciences millet institute, wherein YM15 be by from
Canada has introduced the wild Green foxtail of anti-nicosulfuron and has hybridized with domestic millet, the anti-nicosulfuron herbicide agent millet obtained
New germ plasm, reagent all can obtain from directly purchasing on the market.
One, anti-nicosulfuron Gene A LS resistance locus checking in YM15
Nicosulfuron is that acetolactate synthestase (ALS) suppresses class herbicide, by suppression acetolactate synthestase
(acetolactate synthase, ALS) activity causes the biosynthesis of branched-chain amino acid in plant to be obstructed, and suppresses cell
Differentiation, thus cause plant growing.The sudden change of conserved positions in ALS avtive spot, frequently can lead to it and removes ALS inhibitor class
Grass agent produces the resistance of varying level.Laplante etc. (2009) nicosulfuron anti-to wild sudden change Green foxtail, research shows
Its resistance is to be sported T by single bases G of 3 ' end avtive spots, causes codon AGC to sport ATC, causes different bright amino
Acid replaces what glycine caused.
In order to verify this resistance locus, the anti-nicosulfuron Green foxtail als gene sequence announced with reference to NCBI
(GenBank accession no.:KF020517) and Yungu No.1 als gene (GenBank accession no.:XM_
004952503.2) sequence, designs primer amplification YM15, weighing apparatus paddy No. 12 and other 4 parts of nicosulfuron sensitivity millet materials short
Peaceful Huang, Ji Gu 19, Henan paddy No. 18, Ji Gu 31,3 ' end ALS sequences of short peaceful Huang, and it is checked order.Sequencing result shows weighing apparatus
No. 12 and 5 parts millet materials of paddy are all G in this site, for not resistance nicosulfuron millet, and in YM15 for T with predict the outcome
Unanimously (such as Fig. 1), illustrate that YM15 is resistance nicosulfuron millet.Illustrate that the sudden change of this site causes it to produce nicosulfuron and resists
Property.By named for this resistant gene NiALS.
Two, Rapid identification millet isozygoty, heterozygosis, the molecular marker of sensitive nicosulfuron herbicide agent gene
Embodiment 1
Rapid identification millet anti-nicosulfuron gene pure, heterozygosis or not anti-method, millet material to be identified is
YM15, weighing apparatus paddy No. 12.
Identify by detecting single base of als gene avtive spot in millet material YM15 to be measured, weighing apparatus paddy No. 12, institute
State the nucleotide sequence of als gene as shown in GenBank accession no.:XM_004952503.2, pass through research design
Amplimer, carries out genomic PCR amplification, and the amplified production after then being expanded by PCR carries out enzyme action, uses agarose gel electrophoresis
Identify:
Inventor is by carrying out restriction endonuclease analysis at NiALS and responsive type ALS base mutation, and result shows
There is PvuII restriction endonuclease recognition sequence CGATCG at NiALS base mutation, and in sensitive gene, this site sequence is
CGAGCG, it is impossible to identified by PvuII.Therefore by research design primer, PCR amplification comprises the fragment in this site, uses PvuII enzyme
When cutting, the banding pattern varied in size by generation, and then differentiation is isozygotied, heterozygosis, Sensitive genotype.If homozygous resistant gene, by
After PvuII enzyme action, produce two banding patterns differed in size；If sensitive gene, then can not cut, banding pattern is single tape；As
Fruit is to there will be after heterozygosis resistance then enzyme action to isozygoty and two kinds of sensitive banding patterns, i.e. three bands.By agarose gel electrophoresis, purple
Outer imager is observed and just can be distinguished.Compared with polyacrylamide gel electrophoresis, agarose gel electrophoresis has convenient, fast, simple
Single feature.
1. the design of amplimer
Owing to NiALS avtive spot is positioned at 3 ' ends, find to design primer in this gene through research, because of fragment after enzyme action
Less cannot distinguish with agarose gel electrophoresis.In order to develop the molecular marker that available agarose gel electrophoresis differentiates, pass through
Research, it is thus achieved that this gene 3 ' holds outer sequence (as shown in sequence table).Designing forward primer with gene template, 3 ' hold outer sequential design
Reverse primer.
Primer sequence is: Nicaps-F:3 '-TGCAAGCAGGGCTAAGATTG；
Nicaps-R:3 '-GTACCCCCTTTCATGCACAA.
The preparation of the genomic DNA template of millet material the most to be measured:
The acquisition of YM15 genomic DNA template: take millet YM15 blade 100-200mg to be measured, extracts gene by CTAB method
Group DNA, obtains YM15 genomic DNA；
The acquisition of weighing apparatus No. 12 genomic DNA template of paddy: take millet to be measured weighing apparatus No. 12 blade 100-200mg of paddy, use CTAB method
Extract genomic DNA, obtain No. 12 genomic DNAs of paddy that weigh.
3. genomic PCR amplification
PCR amplification is carried out by following reaction system and response procedures:
PCR reaction system is as shown in table 1:
Table 1
| 2 × PCR mix (Quan Shijin) | 10μl |
| Nicaps-F | 0.5μl |
| Nicaps-R | 0.5μl |
| DNA profiling | 1μl |
| ddH2O | 8μl |
| Cumulative volume | 20μl |
Response procedures is: 94 DEG C of denaturations 4min, 94 DEG C of degeneration 40s, and 57 DEG C of annealing 30s, 72 DEG C extend 80s, and 35 are followed
Ring, 72 DEG C of insulation 7min.
Pcr amplification product such as Fig. 2, pcr amplification product fragment length is 1300bp.
4. enzyme action is identified
Take 7 μ lPCR amplified productions, use PvuI digestion verification.Enzyme action system is as shown in table 2:
Table 2
, there is the single slice of 1293bp, weighing apparatus is described in enzyme action qualification result such as Fig. 3 after weighing apparatus No. 12 pcr amplification product enzyme action of paddy
Paddy No. 12 is the millet of responsive type (not resisting) nicosulfuron, produces 804bp and 498bp size 2 after YM15PCR amplified production enzyme action
Band, stripe size is consistent with expection, illustrates that YM15 is the millet of anti-nicosulfuron of isozygotying.Illustrate that this labelling can be used for distinguishing
Anti-, sensitive nicosulfuron material.By named for this labelling NiALS-CAPs.
5. verify
Direct sequencing is verified: by detecting als gene the 1877th in millet material YM15 to be measured and weighing apparatus paddy No. 12
Base is identified, the nucleotide sequence of described als gene such as GenBank accession no.:XM_004952503.2 institute
Show, comprise the following steps:
A, tentatively discriminating are anti-, the most anti-nicosulfuron millet material；By direct Sequencing, detect in millet material YM15 to be measured
The 1877th deoxyribonucleotide of als gene is T, illustrates that millet material YM15 to be measured is resistance nicosulfuron millet material；
Detecting the 1877th deoxyribonucleotide of als gene in millet material to be measured weighing apparatus paddy No. 12 is G, illustrates that millet material to be measured weighs
Paddy No. 12 is not resistance nicosulfuron millet material；
B, anti-, the discriminating of heterozygosis anti-nicosulfuron millet material of isozygotying: will detect millet material YM15 to be measured, by surveying
Sequence set peak is observed, and order-checking peak value is unimodal, illustrates that millet material YM15 to be measured is homozygous resistant nicosulfuron millet material.
It is correct for demonstrating this method by direct sequencing, and millet material YM15 to be measured is homozygous resistant nicosulfuron
Millet material, millet material to be measured, weighing apparatus paddy No. 12 is not resistance nicosulfuron millet material.
Embodiment 2, the NiALS-CAPs checking in F2 colony
In order to verify the feasibility of this labelling further, detection weighing apparatus paddy No. 12 × YM15F2 plant population.F2 plant is at Seedling
Phase herbicide spraying.Therefore detection plant should be and isozygotys or heterozygosis resistant plant.PCR amplification and enzyme action are ibid.Detect 68 strains altogether,
Partial detection such as Fig. 4.21 strains that have of two bands occur in 68 strains, three bands have 47 strains, a band does not occurs.Root
Being homozygous resistant plant according to labelling principle two band, three bands are heterozygosis resistant plant, single slice for responsive type because seedling stage
This colony herbicide spraying, therefore sensitive kinds banding pattern does not occurs, testing result is consistent with expection, partial detection such as Fig. 4.Separately
Outer two band plant numbers and a band plant number ratio are 2:1 (P=0.7641, χ2=0.0901) mendelian inheritance, is met fixed
In rule, F2 plant is isozygotied and manifests: heterozygosis: the law of homozygous dominant=1:2:1.Further relate to this Marker Identification result accurately and reliably.
Take respectively and amplify three bands and two strip portion plant DNA send to order-checking, according to the verification mark inspection of order-checking set peak
The accuracy surveyed.What sequencing result showed single slice is unimodal at mutational site, for homozygous resistant；And the order-checking peak of three bands
Value is set peak, for heterozygosis resistance.Consistent with marker detection result.Illustrate that this labelling can accurately detect anti-, the heterozygosis of isozygotying anti-and right
The gene type that nicosulfuron is sensitive, result is accurately and reliably.
Embodiment 3, the NiALS-CAPs detection in different cultivars
In order to verify specificity and the imALS-CAPs labelling adaptability of primer, have detected 50 parts of paddy from different regions
Sub-kind.Result show this primer in 50 parts of materials the most amplifiable go out purpose size specific band, pcr amplification product PvuI
Enzyme action result is single slice, consistent with expected results.Partial detection such as Fig. 5.Illustrate that this labelling can be used for planted in different ecological areas
Territory, the molecular marker of different cultivars anti-nicosulfuron millet selection-breeding, fast accurate identifies that anti-, the heterozygosis of isozygotying is anti-and responsive type is planted
Strain, thus accelerate the selection-breeding that millet nicosulfuron is anti-, feel the supporting kind of homotype Sister Lines, improve breeding efficiency.
In sum, excavate and utilize the Novel herbicide resistance gene that can be used for Millet Breeding, resisting for widening millet
Herbicide breeding, increase millet Herbicid resistant multiformity significant.The initiative of anti-nicosulfuron millet kind matter is
Cultivate new herbicides millet to lay a good foundation.This research is according to list base prominent at anti-nicosulfuron Gene A LS avtive spot
Become, establish that qualification accurate, simple and quick is anti-, sense, the method for heterozygosis nicosulfuron millet.With conventional chambers culture dish and field
Plant identification method is compared, and it is reliable and stable that the method has result, advantage easy and simple to handle, quick, automaticity is high.By this
Molecular mark, can accelerate the cultivation that anti-nicosulfuron millet is anti-, feel the supporting kind of homotype Sister Lines, shorten breeding week
Phase, improve breeding efficiency.
Claims (9)
1. Rapid identification millet anti-nicosulfuron gene pure, heterozygosis, the method for the most anti-type, it is characterised in that treated by detection
Survey the base of als gene the 1877th in millet material to identify, comprise the following steps:
A, genomic PCR amplification: use amplimer with the genomic DNA of millet material to be measured as template, carry out PCR amplification,
Obtain pcr amplification product；
B, enzyme action process: the pcr amplification product obtained with digestion with restriction enzyme, obtain digestion products fragment；
C, the qualification of anti-nicosulfuron gene: check digestion products fragment by agarose gel electrophoresis, if after enzyme action being wall scroll
Band, millet material the most to be measured is the most anti-nicosulfuron millet material；If after enzyme action being two bands, millet material the most to be measured is for isozygotying
Anti-nicosulfuron millet material；Being three bands after enzyme action, millet material the most to be measured is heterozygosis anti-nicosulfuron millet material.
Rapid identification millet the most according to claim 1 anti-nicosulfuron gene pure, heterozygosis, the method for the most anti-type, its
Being characterised by, the Primer of described amplimer is as follows with nucleotide sequence:
Nicaps-F:3 '-TGCAAGCAGGGCTAAGATTG；
Nicaps-R:3 '-GTACCCCCTTTCATGCACAA.
Rapid identification millet the most according to claim 1 anti-nicosulfuron gene pure, heterozygosis, the method for the most anti-type, its
Being characterised by, described restricted enzyme is PvuI.
Rapid identification millet the most according to claim 1 anti-nicosulfuron gene pure, heterozygosis, the method for the most anti-type, its
Being characterised by, the genomic DNA of millet material to be measured is obtained by following method: takes millet blade 100-200mg to be measured, uses CTAB
Method extracts genomic DNA.
Rapid identification millet the most according to claim 1 anti-nicosulfuron gene pure, heterozygosis, the method for the most anti-type, its
Being characterised by, in step A genomic PCR amplification, PCR reaction system is: 2 × PCR mix, 10 μ l；Nicaps-F, 0.5 μ l；
Nicaps-R, 0.5 μ l；Millet genomic DNA template to be measured, 1 μ l；ddH2O, 8 μ l；Cumulative volume, 20 μ l.
Rapid identification millet the most according to claim 1 anti-nicosulfuron gene pure, heterozygosis, the method for the most anti-type, its
Being characterised by, in step A genomic PCR amplification, PCR response procedures is: 94 DEG C of denaturations 4min, 94 DEG C of degeneration 40s, and 57 DEG C are moved back
Fire 30s, 72 DEG C extend 80s, 35 circulations, 72 DEG C of insulation 7min.
Rapid identification millet the most according to claim 1 anti-nicosulfuron gene pure, heterozygosis, the method for the most anti-type, its
Being characterised by, during step B enzyme action processes, enzyme action system is: pcr amplification product, 7 μ l；10 × buffer, 1 μ l；PvuI, 0.2 μ
l；ddH2O, 1.8 μ l；Cumulative volume, 10 μ l, 37 DEG C of enzyme action 30min.
8. Rapid identification millet anti-nicosulfuron gene pure, heterozygosis, the test kit of the most anti-type, it is characterised in that: described reagent
Box includes Rapid identification millet anti-nicosulfuron gene pure, heterozygosis or the reagent of non-resistance, and described reagent is by SEQ ID
The amplified production of the DNA molecular composition shown in NO.1.
9. the application in identifying millet nicosulfuron character to be measured of the test kit described in claim 8.
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| CN106376454A (en) * | 2016-09-13 | 2017-02-08 | 河北省农林科学院谷子研究所 | Breeding method of anti-nicosulfuron millet germplasm and application |
| CN106376454B (en) * | 2016-09-13 | 2018-12-25 | 河北省农林科学院谷子研究所 | The selection of anti-nicosulfuron millet germplasm and application |
| CN106636336A (en) * | 2016-10-12 | 2017-05-10 | 河北省农林科学院谷子研究所 | Method for rapidly and accurately identifying homozygosis, heterozygosis and non-resistance types of sethoxydim-resistant gene of foxtail millet |
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