CN110029190A - A kind of cabbage type rape Drought-tolerant gene and its molecular labeling and application - Google Patents
A kind of cabbage type rape Drought-tolerant gene and its molecular labeling and application Download PDFInfo
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- CN110029190A CN110029190A CN201910446222.9A CN201910446222A CN110029190A CN 110029190 A CN110029190 A CN 110029190A CN 201910446222 A CN201910446222 A CN 201910446222A CN 110029190 A CN110029190 A CN 110029190A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a kind of cabbage type rape gene and its applications.The present invention has screened one and has had functional drought tolerance related gene B nA.NF-YA7 in rape different growing, go out drought-enduring material according to the dCAPS label energy precise Identification of BnA.NF-YA7 gene SNP site design, there is certain versatility, eurytopicity, timeliness.It is able to carry out the identification of cabbage type rape drought tolerant germplasm resource and molecular marker assisted selection, is laid a good foundation for breeding is drought-enduring and agronomy, quality trait are excellent breeding material or kind.
Description
Technical field
The present invention relates to a kind of Drought-tolerant gene of cabbage type rape and its molecular labeling and applications.
Background technique
The drought tolerance phenotype of crop be by itself physiological resistance and architectural characteristic and growth and development dynamics rhythm with
What the degree of agroclimate factor variation cooperation determined, with crop species, material genotype, morphological characters, physiological and biochemical procedure
And arid generation period, intensity and length of time are related, are a complicated Comprehensive Traits.
The most commonly used is crop field Direct Identifications for Crop Drought Resistance identification technology, furthermore also have non-irrigated canopy control water to identify and control soil
The basin alms bowl of earth moisture content is tested.Identification of indicator mainly includes yield, form, Physiological and biochemical index etc., furthermore also according to more
Item index institute's measured data obtains the drought-enduring total class value of material or using the Subordinate Function expert evidence drought tolerance in fuzzy mathematics.
Crop field Direct Identification method is only limitted to be identified in arid, semiarid zone or arid season, be limited by external environment；And non-irrigated canopy
It identifies with basin alms bowl and is limited by facility space, be unfavorable for extensive material in the same period progress drought tolerance mirror under i.e. same environment
It is fixed.In addition, carrying out identification to single index lacks accuracy, and to multiple indexs carry out evaluation program complicated, time-consuming, work consuming and
There is destructiveness to material.
Rape is the important oil crops in China, the cabbage type rape of current main cultivation entire season of growth water requirement compared with
Greatly, poor to the adaptability of arid.The drought tolerance of cabbage type rape is by controlled by multiple genes and vulnerable to such environmental effects
Complicated Comprehensive Traits, but in recent years, research of the cabbage type rape in terms of drought stress also focuses primarily upon QTL, significant association
The excavation of label and candidate gene does not obtain the allele with significant drought tolerance at present, it is auxiliary to be not used to molecular labeling
Help the application selected in terms of germplasm identification and drought-enduring variety breeding.
During the identification of cabbage type rape drought tolerant germplasm resource and breeding of new variety, general survey method is mainly used,
And general survey method work consuming is time-consuming, efficiency is lower, has destructiveness to material, vulnerable to external environment and subjective consciousness shadow
Sound, accuracy are poor, seriously constrain the breeding of drought tolerance kind.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of Drought-tolerant gene of cabbage type rape and its molecular labeling with
Using, gene of the present invention can all be induced to express in sprouting stage, seedling stage and maturity period, and with the allele
Material all has stronger drought tolerance in sprouting stage, seedling stage and maturity period.The present invention utilizes the molecule mark of allele exploitation
Note, can by molecular markers for identification germ plasm resource drought tolerance and assist-breeding drought tolerance new varieties, be not easy it is affected by environment, accurately
Property it is high；It only needs to extract DNA using a little blade of plant, it is small to breeding material destructiveness；It can be selected in early days, efficiency
Height is conducive to the process for accelerating to cultivate drought-enduring, high yield and fine quality.
Technical solution used by the present invention solves the above problems is:
The Drought-tolerant gene of cabbage type rape, the gene be cabbage type rape A09 chromosome a nuclear factor etc.
Position gene, being located at the base at 19159584bp is C.
Cabbage type rape gene BnA.NF-YA7 of the present invention is (positioned at a core of cabbage type rape A09 chromosome
Transcription factor) allele, be located at A09 chromosome 19158540bp-19160295bp, specifically, be located at 19159584bp
Base be C.In cabbage type rape gene order disclosed in the prior art, the base at 19159584bp is G, the present invention
It is different from sequence disclosed in existing cabbage type rape.Further, in the prior art, it there are no the equipotential of the position gene order
Gene is used for the related of cabbage type rape drought resisting screening varieties and records, and existing sequence cannot be used for identifying or identify or
Person confirms whether the cabbage type rape rape belongs to drought-resistant variety, and sequence of the invention can be used in identifying or identify or
Confirm whether the cabbage type rape rape belongs to drought-resistant variety.
Further, the gene is located at the sequence of cabbage type rape A09 chromosome 19159555bp-19159690bp,
With sequence shown in SEQ ID NO:1.
Said gene sequence is in the application in screening, breeding, preparation or detection cabbage type rape drought-enduring variety.
Above-mentioned sequence answering in screening, breeding, preparation or detection cabbage type rape drought-enduring variety as molecular labeling
With.
Specifically, as cabbage type rape have above-mentioned gene order, then be drought-enduring type cabbage type rape.
A kind of screening, breeding, preparation or the detection method of cabbage type rape drought-enduring variety, include the following steps:
1) it is located at the base position of 19159584bp based on cabbage type rape A09 chromosome, utilizes dCAPS Finder
2.0 online website design primers, in conjunction with restriction enzyme NdeI exploitation dCAPS label；
2) DNA of multiple strains is extracted, sequencing judgement is then drought-enduring if cabbage type rape has above-mentioned gene order
Type cabbage type rape.
Specifically, in the step 1), the base that cabbage type rape A09 chromosome is located at 19159584bp is the gene the
SNP on three exons.
Specifically, in the step 2), left and right primer sequence used in PCR amplification is carried out after extracting DNA are as follows:
Left primer 5'-CCATATCCAGATCCTTACTACAGACATAT-3'；
Right primer 5'-TCCCATTAAATGTGCATGTACCTGA-3'.
In the step 2), extracts the laggard row of DNA and carry out PCR amplification, digestion and electrophoresis, gel imaging is used after the completion of electrophoresis
System observation, if the band of display is located at 105bp, for drought-enduring material.
The PCR amplification program is as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of renaturation
1min, 35 circulations；72 DEG C of extension 10min, 12 DEG C of preservations.
The pcr amplification reaction system is as follows: 12.5 μ L 2 × Taq Master Mix buffers, the primer of 10 μm of ol/L
0.25 μ L, the ddH2O distilled water of Easy Taq enzyme of each 0.5 μ L, 50~100ng genomic DNA, 5U/ μ L complements to 25 μ L；
The pcr amplification product uses 1.8% agarose gel electrophoresis electrophoresis detection, 5.0 μ L of applied sample amount；Electrophoretic buffer
For 0.5 × TAE, 120V electrophoresis 25min；It is observed after the completion of electrophoresis with gel imaging system.
In conclusion the beneficial effects of the present invention are:
1) present invention has screened the equipotential base for having functional drought tolerance related gene in rape different growing
Because of sequence.
2) it is marked according to the dCAPS of cabbage type rape one drought-enduring allelic sequences SNP site design, in conjunction with the present invention
The Drought-tolerant gene of screening, judges whether the plant has the Drought-tolerant gene, and then energy precise Identification goes out whether the plant is drought-enduring
Material has certain versatility, eurytopicity, timeliness.It is able to carry out the identification of cabbage type rape drought tolerant germplasm resource and molecule
Marker assisted selection is laid a good foundation for breeding is drought-enduring and agronomy, quality trait are excellent breeding material or kind.
3) gene order according to the present invention and molecular labeling can be widely applied to cabbage type rape screening, breeding, preparation
Or detection cabbage type rape drought-enduring variety.
Detailed description of the invention
Fig. 1 is the association analysis (a:GP of cabbage type rape genetic mutation Yu cabbage type rape drought tolerance；b:GI；C: chain
Imbalance analysis (LD))；
Fig. 2 is that (a:BnA.NF-YA7 is (positioned at sweet for GP and the GI comparative analysis of different haplotypes under 20%PEG6000 stress
One nuclear factor of blue type rape A09 chromosome) haplotype analysis；The case figure of b:5 kind haplotype material GI；C:5 kind list
The case figure of times profile material GP；Germination performance of the d:5 kind haplotype material under PEG processing；The GP and GI of e:5 kind haplotype material
Statistical chart (p < 0.05))；
Fig. 3 is that different haplotype material drought tolerances compare (after a:20%PEG6000 is handled 7 days under 20%PEG6000 stress
The drought-enduring sex expression of 5 kinds of haplotype materials；The survival rate of 5 kinds of haplotype materials after b:20%PEG6000 is handled 7 days；C:20%
PEG 6000 coerces the dynamic change of lower 5 kinds of haplotypes material blade water content)；
Fig. 4 is that the lower 5 kinds of haplotypes difference strain drought tolerance of drought stress compares that (a: 5 kinds of haplotypes are not after Osmotic treatment 3 weeks
The drought-enduring sex expression of homophyletic based material；B: the Physiological Analysis (POD, SOD and CAT) of 5 kinds of haplotype materials after Osmotic treatment；C: dry
The ABA content of 5 kinds of haplotype materials after drought processing；D: the soluble protein and proline of 5 kinds of haplotype materials after Osmotic treatment
Content；E: the lower 5 kinds of haplotypes material root/shoot ratio of drought stress；F: the MDA content of 5 kinds of haplotype materials after Osmotic treatment；G: dry
Drought coerces lower 5 kinds of haplotype material blades relative water content)；
Fig. 5 is different haplotype material dCAPS label performances.
Specific embodiment
It is of the invention to filter out a kind of cabbage type rape Drought-tolerant gene BnA.NF-YA7 and (be located at cabbage type rape A09 to dye
One nuclear factor of body) allele, be located at A09 chromosome 19158540bp-19160295bp be specifically located at
The base of 19159584bp is C.
Specifically, the drought-enduring allele of the cabbage type rape has nucleotide sequence shown in SEQ ID NO.1.
Said gene sequence and molecular labeling can be used in screening, breeding, preparation or detection cabbage type rape drought-enduring variety；
Its concrete application method are as follows: be then drought-enduring type cabbage type rape if cabbage type rape has above-mentioned gene order.
The present invention will now be described in further detail with reference to the accompanying drawings and the accompanying drawings, but embodiments of the present invention
It is without being limited thereto.
Embodiment 1: percentage of seedgermination (GP) is associated with germination index (GI) full-length genome under cabbage type rape drought stress
Analysis
520 parts of germ plasm resources used in the present embodiment are China's rapeseed breeding and R&D institution's collected both.Every part
Germ plasm resource is chosen 50 full seeds and is carried out disinfection processing using 4%NaClO solution, and the 0.9cm for being covered with filter paper is homogeneously disposed in
In diameter Petri dishes.For the first time, 30ml 20%PEG6000 solution is added in drought stress processing, and 30ml ddH is added in control2O2, it
15ml 20%PEG6000 solution is added in drought stress processing afterwards, and 15ml ddH is added in control2O2, continue to the 7th day.It carries out 3 times
It repeats to test, investigation statistics germination percentage (GP) and germination index (GI).It is based in conjunction with the genotype data that 60K chip analysis obtains
MLM model is associated analysis, obtain under drought stress with germination percentage (GP) and germination index (GI) significantly associated SNP.And
The relevant candidate of screening drought tolerance is further predicted according to decaying section and arabidopsis gene functional annotation where significant association SNP
Gene.
Under table 1BnA.NF-YA7 gene polynorphisms site and its three kinds of analysis models with the lower 181 parts of wild cabbages of drought stress
The association analysis result of type rape seed GP and GI
Percentage of seedgermination (GP) and the analysis of germination index (GI) candidate gene association under cabbage type rape drought stress: such as table
Shown in 1, sequence is resurveyed based on full-length genome as a result, in BnA.NF-YA7 gene internal and promoter region (upstream from start codon
18 SNP 2kb) are detected altogether.It is based on not according to 181 parts of material GP and GI phenotypic datas under these SNP data and PEG processing
Be associated analysis with analysis model, SNP (S9_19159584) to the GP of seed under drought stress and GI it is significant it is related (p <
0.01, Fig. 1 a, b).Meanwhile it being found when carrying out linkage disequilibrium (LD) analysis to this 18 SNP, S9_19159584 and gene
The LD value of internal SNP is lower, higher (Fig. 1 c) with the LD value of the SNP of promoter region.These are the result shows that BnA.NF-YA7 base
There is hereditary variation relevant to the drought-enduring difference of cabbage type rape because interior, i.e. S9_19159584 and cabbage type rape drought tolerance are closed
Connection.
Embodiment 2: haplotype analysis and drought tolerance phenotypic evaluation:
18 SNP detected according to sequence is resurveyed carry out haplotype analysis, obtain altogether 5 main haplotypes (Hap,
Haplotype) (Fig. 2 a) compares GP and GI of these haplotypes under drought stress, as a result, it has been found that the GP and GI of Hap4 will be shown
It writes and is higher than Hap3 and Hap5 (p < 0.05), Hap1 is only second to Hap4, and Hap2 belongs to intermediate form (Fig. 2 b, c).Utilize 20%
PEG6000 simulation stress, the different strains for choosing 5 kinds of haplotypes are verified, the results showed that different under PEG6000 processing
The GP and GI of haplotype material are consistent with early-stage study result, the equal highest of seed GP and GI of Hap4, are secondly Hap1, and Hap3
With GP and GI lower (Fig. 2 d, e) of the Hap5 haplotype material under PEG6000 processing.In addition, more different haplotype materials
Drought tolerance in seedling stage the result shows that, (Fig. 3 a) or natural drought either under 20% PEG6000 simulating drought environment
Under environment (Fig. 4 a), different haplotype material seedling stage drought tolerance results and germination performance are consistent.20%PEG6000 simulation is dry
Under the conditions of drought, the survival rate of Hap4 and Hap1 haplotype material is still up to 87.5% and 56.5% respectively after handling 7 days, and
The survival rate of Hap2 and Hap3 haplotype material is only 10.25% and 12.5% (Fig. 3 a, b).Leaf water content measurement result is aobvious
Showing, PEG6000 processing 1h starts, and 5 kinds of haplotype material blade water content sharply decline, and 3h reaches minimum after stress, this
Rear blade water content gradually starts to restore, Hap4 and Hap1 haplotype material restores very fast, containing before having been approached processing in 12h
Water, and other 3 kinds of haplotype materials restore (Fig. 3 c) completely yet until 72h, show Hap1 and Hap4 compared with Hap2, Hap3 and
Hap5 can comparatively fast adapt to the variation of extraneous osmotic potential.In addition, potted plant experiment the result shows that Continuous Drought handle 3 weeks after, Hap4
Stronger drought tolerance (Fig. 4 a) is shown with the more other 3 kinds of haplotype materials of Hap1 haplotype material.Physiological measurement shows 5 kinds
Apparent difference is presented in haplotype material after Osmotic treatment.After Osmotic treatment, the proline of Hap4 haplotype material and solvable
Property protein content is all remarkably higher than other haplotype materials, especially Hap3.On the contrary, SOD, CAT of Hap4 haplotype material and
MAD content is substantially less than Hap3, ABA content no significant difference.Root/shoot ratio measurement result shows, the Hap1 of drought-enduring type and
The root/shoot ratio of Hap4 shows drought susceptible haplotype material to drought stress significantly less than drought susceptible Hap2, Hap3 and Hap5
Extreme sensitivity, aerial growth is suppressed after being coerced, and drought-enduring haplotype material well developed root system, while aerial part
Normal growth, thus the root/shoot ratio of drought-enduring type is caused to be less than not drought-enduring type.In addition, leaf r elative water content measurement result is aobvious
Show that the leaf r elative water content of the Hap1 and Hap4 of drought-enduring type are significantly higher than drought susceptible Hap2, Hap3 and Hap5 (Fig. 4 b-
g).The above results further confirm, have under drought condition, between 18 SNP difference haplotypes of BnA.NF-YA7 gene apparent
Drought-enduring sex differernce.The difference of two kinds of haplotypes Hap3 and Hap4 of extreme phenotype are concentrated mainly on intragenic 10 SNP.10
There are 2 SNP to be located at UTR region in a SNP, 5 are located on introne, and 3 SNP are only existed on exon.The test of embodiment 1
The result shows that wherein the SNP (S9_19159584) on the 3rd exon is associated with cabbage type rape drought tolerance, i.e. SNP
(S9_19159584) 5 kinds of haplotypes accurately can be divided into drought-enduring and not drought-enduring two kinds of different types (Fig. 2 a).
Embodiment 3: drought tolerance identification is carried out using BnA.NF-YA7 targeting dCAPS molecular labeling
A kind of screening technique of cabbage type rape drought-enduring variety, includes the following steps:
1) based on the base position for being located at 19159584bp based on cabbage type rape A09 chromosome, dCAPS is utilized
The online website design primer of Finder 2.0, in conjunction with restriction enzyme NdeI exploitation dCAPS label；
2) DNA for extracting multiple strains, the gene after following identifications as described in cabbage type rape has SEQ ID NO.1
Sequence is then drought-enduring type cabbage type rape.
Specifically, it in the step 2), extracts the laggard row of DNA and carries out PCR amplification, digestion and electrophoresis, PCR amplification program is such as
Under: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of renaturation 1min, 35 recycle；72 DEG C of extensions
10min, 12 DEG C of preservations.Pcr amplification reaction system is as follows: 12.5 μ L2 × Taq Master Mix buffers, and 10 μm of ol/L's draws
0.25 μ L, the ddH2O distilled water of Easy Taq enzyme of each 0.5 μ L, 50~100ng genomic DNA of object, 5U/ μ L complements to 25 μ L.Expand
Increase production object and uses 1.8% agarose gel electrophoresis electrophoresis detection, 5.0 μ L of applied sample amount；Electrophoretic buffer is 0.5 × TAE,
120V electrophoresis 25min；It is observed after the completion of electrophoresis with gel imaging system, if the band of display is located at 105bp, there is SEQ
Gene order shown in ID NO.1 is drought-enduring material.
Specifically, nucleotide sequence such as SEQ ID NO.2 and SEQ ID NO.3 used in PCR amplification is carried out after extracting DNA
It is shown, i.e. primer sequence are as follows: 5'-CCATATCCAGATCCTTACTACAGACATAT-3' and 5'-
TCCCATTAAATGTGCATGTACCTGA-3'。
Extract the DNA of 5 kinds of multiple strains of haplotype material in embodiment 2, by the present embodiment method progress PCR amplification,
Digestion and electrophoresis, electrophoresis are observed with gel imaging system after the completion, the results show that Hap4 and Hap1 has SEQ ID NO.1 institute
The band of the gene order shown, display is located at 105bp, is drought-enduring material.
The result shows that the dCAPS label that the present invention develops isolates with phenotype and can correctly be divided into 5 kinds of haplotypes resistance to
Non-irrigated and not drought-enduring two class (Fig. 5).
As described above, can preferably implement the present invention.
SEQUENCE LISTING
<110>Southwest University
<120>a kind of cabbage type rape Drought-tolerant gene and its molecular labeling and application
<130>a kind of cabbage type rape Drought-tolerant gene and its molecular labeling and application
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 136
<212> DNA
<213>cabbage type rape
<400> 1
ccatatccag atccttacta cagaagtatc tttgcaccaa acccacaagc gtatccacca 60
cgaccttatg aaaccggggt atggtgtatt acaacgattt cttgatttgg atcatgttaa 120
cagcattttt cacatc 136
<210> 2
<211> 29
<212> DNA
<213>artificial sequence
<400> 2
ccatatccag atccttacta cagacatat 29
<210> 3
<211> 25
<212> DNA
<213>artificial sequence
<400> 3
tcccattaaa tgtgcatgta cctga 25
Claims (10)
1. a kind of Drought-tolerant gene of cabbage type rape, which is characterized in that the gene is a core of cabbage type rape A09 chromosome
The allele of transcription factor, the base positioned at 19159584bp are C.
2. the Drought-tolerant gene of cabbage type rape according to claim 1, which is characterized in that the characteristic sequence position of the gene
In cabbage type rape A09 chromosome 19159555bp-19159690bp, there is sequence shown in SEQ ID NO:1.
3. gene order described in claim 1 or claim 2 is drought-enduring in screening, breeding, preparation or detection cabbage type rape
Application in kind.
4. molecular labeling described in claim 1 or claim 2 is drought-enduring in screening, breeding, preparation or detection cabbage type rape
Application in kind.
5. a kind of screening, breeding, preparation or the detection method of cabbage type rape drought-enduring variety, which is characterized in that including walking as follows
It is rapid:
1) it is located at the base position of 19159584bp based on cabbage type rape A09 chromosome, it is online using dCAPS Finder2.0
Website design primer, in conjunction with restriction enzyme NdeI exploitation dCAPS label；
2) DNA for extracting multiple strains, the gene order as described in cabbage type rape has claim 1 or claim 2, then
For drought-enduring type cabbage type rape.
6. screening, breeding, preparation or the detection method of cabbage type rape drought-enduring variety according to claim 5, feature
It is, in the step 2), carries out left and right primer sequence used in PCR amplification after extraction DNA and be respectively as follows:
Left primer: 5'-CCATATCCAGATCCTTACTACAGACATAT-3'；
Right primer: 5'-TCCCATTAAATGTGCATGTACCTGA-3'.
7. screening, breeding, preparation or the detection method of cabbage type rape drought-enduring variety according to claim 6, feature
It is, in the step 2), extracts the laggard row of DNA and carry out PCR amplification, digestion and electrophoresis, electrophoresis is after the completion with gel imaging system
Overall view is examined, if the band of display is located at 105bp, for drought-enduring material.
8. screening, breeding, preparation or the detection method of cabbage type rape drought-enduring variety according to claim 6, feature
It is, the PCR amplification program is as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of renaturation 1min,
35 circulations；72 DEG C of extension 10min, 12 DEG C of preservations.
9. the screening technique of cabbage type rape drought-enduring variety according to claim 6, which is characterized in that the PCR amplification
Reaction system is as follows: 12.5 μ L 2 × Taq Master Mix buffers, each 0.5 μ L, 50~100ng base of the primer of 10 μm of ol/L
Because of a group DNA, 0.25 μ L, the ddH2O distilled water of Easy Taq enzyme of 5U/ μ L complements to 25 μ L.
10. the screening technique of cabbage type rape drought-enduring variety according to claim 6, which is characterized in that the PCR amplification
Product is detected using 1.8% agarose gel electrophoresis, 5.0 μ L of applied sample amount；Electrophoretic buffer is 0.5 × TAE, 120V electrophoresis
25min；It is observed after the completion of electrophoresis with gel imaging system.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113005217A (en) * | 2021-04-01 | 2021-06-22 | 中国农业科学院生物技术研究所 | ZmNF-YA8 gene SNP marker related to salt tolerance of corn and application thereof |
| CN114672584A (en) * | 2022-04-22 | 2022-06-28 | 西南大学 | Molecular marker primer pair, drought-tolerant cabbage type rape identification method and breeding method |
| CN117106802A (en) * | 2023-09-15 | 2023-11-24 | 西部(重庆)科学城种质创制大科学中心 | Cabbage type rape high-dehiscence-angle resistance gene and identification and application thereof |
| CN117106802B (en) * | 2023-09-15 | 2024-04-30 | 西部(重庆)科学城种质创制大科学中心 | Cabbage type rape high-dehiscence-angle resistance gene and identification and application thereof |
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| CN117106802B (en) * | 2023-09-15 | 2024-04-30 | 西部(重庆)科学城种质创制大科学中心 | Cabbage type rape high-dehiscence-angle resistance gene and identification and application thereof |
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