CN108728563A - The InDel of Chinese cabbage Bra013400 frameshift mutations is marked and its application in the practices of breeding - Google Patents
The InDel of Chinese cabbage Bra013400 frameshift mutations is marked and its application in the practices of breeding Download PDFInfo
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Abstract
The invention discloses a kind of codominant marker of the encoding gene Bra013400 wild types and mutant of identification Chinese cabbage TNL-WRKY, the codominant marker includes：With wild type relevant label Bra013400W, clip size 140bp, as shown in SEQ ID No.1；With mutant relevant label Bra013400m, size 135bp, as shown in SEQ ID No.2.Present invention obtains wild types and mutant that one is located at the gene Bra013400 that TNL-WRKY may be encoded on A01 chromosomes, and develop the codominant marker of identification wild type and mutant.The label can be used for screening Chinese cabbage natural population, the detection means of the disease-resistant R gene locis of efficient Chinese cabbage is provided, the material for the carrying wild type gene that codominant marker through the invention detects can be as the important materials of breeding for disease resistance, for cultivating disease-resistant New Chinese Cabbage Variety.
Description
Technical field
The present invention relates to vegetable breeding technical fields, and in particular to a kind of InDel of Chinese cabbage Bra013400 frameshift mutations
Label and its application in the practices of breeding.
Background technology
There is abundant disease-resistant gene (Resistance gene) i.e. R genes in Plant Genome, to ensure it in life
The whole cycle of long development is resisted miscellaneous pathogenic microorganism and is attacked.The R genes cloned so far have had more than 100, grind
Study carefully discovery：One major class R gene encoding productions generally comprise Toll/Interleukin-1 receptor similar structures (Toll/
Interleukin-1 receptor, TIR), nucleotide binding site (Nucleotide binding site, NBS) and rich in bright
Propylhomoserin repeats (Leucine-rich repeats, LRRs) etc..For specific R genes, often above-mentioned several motifs
The combination of one or more of motifs.According to the motif that R genes are included, R genes are roughly divided into eight class (Gururani, MA；
Venkatesh,J；Upadhyaya,CP；Nookaraju,A；Pandey,SK；Park,SW.2012.Plant disease
resistance genes:Current status and future directions.Physiological and
Molecular Plant Pathology.78:51-65.), i.e. TIR-NBS-LRR (abbreviation TNL classes), CC-NBS-LRR, LRR-
TrD, LRR-TrD-Kinase, TrD-CC, TIR-NBS-LRR-NLS-WRKY, LRR-TrD-PEST-ECS and Enzymatic R-
genes.It has been reported that R genes in, disease-resistant spectrum is very extensive, the pathogenies object such as existing directed toward bacteria, fungi and mastigomycetes
, also there is the resistance for virus.Although specific R genes are just for specific pathogeny effector, since R genes are with dominant
Or the heredity of semidominant mode, and it is large number of, thus compared with recessive gene for, this makes it in breeding work using more convenient
(Staskawicz,B.J.,Ausubel,F.M.,Baker,B.J.,Ellis,J.G.,and Jones,
J.D.G.1995.Molecular genetics of plant disease resistance.Science.268:661-
667.)。
Chinese cabbage full-length genome has been sequenced, and to its potential R gene carried out comprehensive annotation (Cheng, F., Liu,
S.,Wu,J.,Fang,L.,Sun,S.,Liu,B.,Li,P.,Hua,W.,Wang,X.2011.BRAD,the genetics and
genomics database for Brassica plants.BMC plant biology.11:136.).In the full base of Chinese cabbage
Because in group strain Chi-fu401 of sequencing, there is 244 total 16 class R genes, wherein most clusters exist.Because of R genes
Meaning is larger in the practices of breeding, has become the hot spot of industry to R gene functional research.Research in recent years is found：C-terminal has
WRKY structural domains and with function of receptors TNL class R genes be effector excitation plant immune disease resistance mechanisms important regulating and controlling
The factor, play a significant role especially in the form of TNL-NLR heterodimers when coping with bacterium, fungal infection (Katharina
Heidrich,KenichiTsuda,Servane Blanvillain-Baufumé,Lennart Wirthmueller,
Jaqueline Bautor and Jane E.Parker.2013.Arabidopsis TNL-WRKY domain receptor
RRS1 contributes to emperature-conditioned RPS4 auto-immunity.Frontiers in
Plant Sciences.2013,4:Pp 1~13).
But there is not yet the report of receptor class TNL-WRKY gene clonings in Chinese cabbage.It is coped in plant in view of TNL-WRKY
Important regulating and controlling effect when pathogen infection, it is necessary to extensive screening is carried out to this kind of R genes in existing Chinese-cabbage Germplasm,
To find each site mutation type that may be present；Simultaneously for the sequence difference in mutational site and wild type, development zone dividing line
The locus specificity molecular labeling of raw type and mutant.
Invention content
For the blank in terms of the R identified for genes with TNL-WRKY structure types in current Chinese cabbage, mesh of the invention
Be to provide Chinese cabbage Bra013400 frameshift mutations InDel label and its application in the practices of breeding.
The present invention obtains one and is located at the gene Bra013400's that may encode TNL-WRKY on A01 chromosomes first
Wild type and mutant, secondly according to the sequence difference of the two, design primer, develops identification wild type and the total of mutant shows
Property marks and is used for marker assisted selection.
To achieve the above object, the present invention adopts the following technical scheme that：
The first aspect of the present invention provides a kind of encoding gene Bra013400 wild types of identification Chinese cabbage TNL-WRKY
With the codominant marker of mutant.
The present invention codominant marker include：With wild type relevant label Bra013400W, clip size 140bp, such as
Shown in SEQ ID No.1；With mutant relevant label Bra013400m, size 135bp, as shown in SEQ ID No.2.
Application of the above-mentioned codominant marker in Chinese-cabbage Germplasm identification and/or breeding for disease resistance is also the guarantor of the present invention
Protect range.
The second aspect of the present invention provides the primer sets for differentiating above-mentioned codominant marker, the sequence of the primer sets
It is specific as follows as shown in SEQ ID No.3 and SEQ ID No.4：
Forward primer Bra013400F：5'-AAGAAATCTTCGAGTCGGAGGG-3'；(SEQ ID No.3)
Reverse primer Bra013400R：5'-AGACGGAAACTAGTCCTATCGGGTA-3'；(SEQ ID No.4)
Above-mentioned primer sets are preparing encoding gene Bra013400 wild types and mutation for identifying Chinese cabbage TNL-WRKY
Application in the kit of body is also protection scope of the present invention.
The third aspect of the present invention provides a kind of for identifying that the encoding gene Bra013400 of Chinese cabbage TNL-WRKY is wild
The kit of raw type and mutant, the kit include：Primer shown in SEQ ID No.3 and SEQ ID No.4.
Application of the mentioned reagent box in Chinese-cabbage Germplasm identification and/or assistant breeding is also the protection model of the present invention
It encloses.
The fourth aspect of the present invention provides a kind of method identified or auxiliary identifies Chinese cabbage cultivar to be measured, including as follows
Step：
(1) PCR amplification is carried out to the genomic DNA of test individual using above-mentioned primer sets；
(2) size of detection amplified fragments contains wild type gene if amplifying the band of 140bp in genome；
If amplifying the band of 135bp, which is mutated.
In step (1), the condition of PCR amplification is：95 DEG C of pre-degenerations 5 minutes；95 DEG C are denaturalized 30 seconds, and 62 DEG C are annealed 30 seconds,
72 DEG C extend 10 seconds, 35-38 cycle；Last 72 DEG C extend 5 minutes.
The material of the carrying wild type gene of above method screening can be as the important materials of breeding for disease resistance, for cultivating
Disease-resistant New Chinese Cabbage Variety.
Above-mentioned technical proposal has the advantages that：
(1) present invention obtains the open countries that one is located at the gene Bra013400 that TNL-WRKY may be encoded on A01 chromosomes
Raw type and mutant, and develop the codominant marker of identification wild type and mutant.The label can be used for screening great Bai
Dish natural population provides the detection means of the disease-resistant R gene locis of efficient Chinese cabbage, codominant marker's detection through the invention
The material of the carrying wild type gene gone out can be as the important materials of breeding for disease resistance, for cultivating disease-resistant New Chinese Cabbage Variety.
(2) codominant marker that the present invention is developed for wild type and mutator, can be used for Chinese-cabbage Germplasm
The backcross transformation assisted Selection of screening and disease-resistant material.The present invention codominant marker application can greatly simplify screening means,
Shorten the transformation time limit, avoids the blindness selected in conventional breeding methods.It, can when cultivating broad-spectrum disease resistance New Chinese Cabbage Variety
To realize marker assisted selection, breeding efficiency is improved.Since this label is inside functional gene, the standard of selection
True rate is up to 100%.Simultaneously also Molecular Detection tool is provided to reappraise the application value of Chinese cabbage resource.
Description of the drawings
The accompanying drawings which form a part of this application are used for providing further understanding of the present application, and the application's shows
Meaning property embodiment and its explanation do not constitute the improper restriction to the application for explaining the application.
Fig. 1：Retrieval result of the target gene Bra013400 coded sequences in NCBI albumen databases；(diagram gives
The meaning of the translator of Chinese of second and third homologous sequence, remaining sequence is similar).
Fig. 2：Primer combines Bra013400F/Bra013400R and is preced in 291 and 06-247 in two parts of typical Chinese cabbage materials
Amplification, M is DNA molecular amount standard 50bp ladder.
Fig. 3：Aim sequence comparison result in two parts of materials.As can be seen from the figure mutant nucleotide sequence Bra013400m is than wild
Type Bra013400W lacks five bases.
Fig. 4：The difference for the translation product that wild type gene and mutant gene derive.Wild type is given in figure A boxes
Codon reading conditions of the base coding region between 200-210bp, it is illustrated that normal reading.Mutation base is given in figure B boxes
Because of codon reading conditions of the coding region between 200-210bp, it is illustrated that frameshift mutation causes to terminate in advance.
Fig. 5：Primer combines Bra013400F/Bra013400R
Detection cases of the F2 derived from parent for part single plant (B).It is self-mating system hat 291 that CK1, which is self-mating system 06-247, CK2, in figure A,
1-10 is ten parts of Chinese cabbage resources, wherein the same CK1 of 1,5,8,10 genotype in the sites Bra013400, is wild type, remaining is same
CK2, gene are mutated；It is hat 291 that P1, which is 06-247, P2, in figure B, and 11-20 is 10 single plants in F2 groups, number
15, the same P1 of 18 genotype in the sites Bra013400, is wild type, and number 11,13,14,17,19 is mutation with parent P2
Body, 12,16,20 be heterozygous.
Specific implementation mode
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another
It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific implementation mode, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation and/or combination thereof.
As background technology is introduced, there has been no about receptor class TNL-WRKY genes gram in Chinese cabbage in the prior art
Grand report.Based on this, the present invention proposes the InDel labels of Chinese cabbage Bra013400 frameshift mutations and its in breeding practice
In application.
The present invention has carried out heavy sequencing analysis using 27 parts of Chinese cabbage core resources of full-length genome weight sequencing technologies pair first.
In mining analysis sequencing data, it is found that the variation of sequence has occurred in a large amount of disease-resistant related genes, one of them is to be located at A01 to contaminate
Bra013400 on colour solid, because on column " nr_annotation " that sequencing file provides, which is disease
Resistant protein, and the annotation provided on the column " Swissprot_annotation " is " Probable WRKY
Transcription factor 52, OS=Arabidopsis thaliana GN=WRKY19 ", in NCBI data, with
The gene coding amino acid sequence homology, rape (Brassica napus) or turnip during highest former sequences are all come
(Brassica rapas) both crops more closely belong to Brassica genus with Chinese cabbage affiliation.As shown in Figure 1, second and third
The annotation provided in homologous sequence is " ill-resistant protein RRS1 isomers X1/X2 " respectively.Further literature search finds quasi- south
The Double albumen of mustard RPS4-RRS1 is the main regulatory factors (Mari for mediating arabidopsis to bacterium and fungi resistance of wide spectrum
Narusaka,Kazuhiro Toyoda,Tomonori Shiraishi1,Satoshi Iuchi,Yoshitaka Takano,
Ken Shirasu&Yoshihiro Narusaka,Leucine zipper motif in RRS1 is crucial for
the regulation of Arabidopsis dual resistance protein complex RPS4/RRS1,2016,
Scientific Reports).There is not been reported for the function of the genoid and its mutant identification in Chinese cabbage, it is therefore necessary to
Screening is carried out to sequence variations situation of the gene in Chinese cabbage resource, to develop the molecule mark for respective mutation type
Note provides assisted Selection to cultivate disease-resistant New Chinese Cabbage Variety using the germ plasm resource for carrying different TNL-WRKY gene types
Tool.
Due to there is various R genes in Chinese cabbage genome, the R genes in each site are possible to resist different pathogenies, due to R
Gene is in dominant inheritance, as long as having the gene, even heterozygous state, then certain material is just resistant to specific pathogeny.If can
Respective R genes wild-type status is identified in different loci respectively, exploitation is detected for respective R genes wild type and mutant
Locus specificity molecular labeling, then can be by convergent cross and binding molecule marker assisted selection, by multiple wild type positions
Point is aggregated in same a material, creates broad-spectrum disease resistance Chinese cabbage new germ plasm.
In one embodiment of the present invention, it is found that wild type material " 06-247 " using special primer clone technology
In gene Bra013400s of the gene Bra013400 for wild-type status in susceptible material " hat 291 " be mutation status, open
The codominant marker of both identifications is sent out, the codominant marker includes：With the relevant label Bra013400W of wild type, segment
Size 140bp, as shown in SEQ ID No.1；With mutant relevant label Bra013400m, size 135bp, such as SEQ ID
Shown in No.2.
Genotype that can be to introgressive line and Chinese-cabbage Germplasm in the site using the label is accurately selected
It selects, to verify Chinese cabbage resource in the sequence situation in the site, and the abundanter disease-resistant material of genetic background can be created.
As a preferred option, the screening process of the label is as follows：
(1) abrupt information provided according to full-length genome weight sequencing data, in sudden change region both sides, conserved regions design is special
Primer；
(2) CTAB methods are used, the genomic DNA of two parts of typical materials is extracted, portion is wild type material " 06-247 ",
Portion is mutant material " hat 291 "；
(3) PCR amplification and PAGE electrophoresis detections are carried out to target area, has obtained two segments of estimated size；
(4) clone and sequence verification are carried out to amplified production respectively, finds to meet with weight sequencing data, the expansion confirmed
Increase production the label that object is exactly required.
(5) PCR amplification and electrophoresis detection further have been carried out to part core resource and a F2 group with gained label,
The practicability of label is demonstrated, and has further confirmed that the variation situation and the label in relation to material in the sites Bra013400
Application in breeding selection.
The codominant marker of the application can be used as molecular labeling to be applied to Chinese-cabbage Germplasm identification and breeding auxiliary
Selection and breeding.In the another embodiment of the application, after selecting the germplasm materials containing wild-type status in 06-247 or transformation
Generation.Concrete application mode is：PCR amplification is carried out to the genomic DNA of test individual using the pair of primers of the codominant marker,
The size for detecting amplified fragments, if amplify 140bp band, if contain wild type gene in genome, expand 135bp's
Band, then the gene is mutated.
Primer sequence is specific as follows：
Forward primer Bra013400F：5'-AAGAAATCTTCGAGTCGGAGGG-3'；(SEQ ID No.3)
Reverse primer Bra013400R：5'-AGACGGAAACTAGTCCTATCGGGTA-3'；(SEQ ID No.4)
It should be noted that due to having not yet to see the report about receptor class TNL-WRKY gene clonings in Chinese cabbage,
Relationship between gene Bra013400 on A01 chromosomes and coding TNL-WRKY is also unknown.System of the present invention is for the first time
It was found that gene Bra013400 may encode TNL-WRKY, and there are sudden change region in Bra013400 genes, and further send out
Now the sudden change region is related to Chinese cabbage disease resistance.It is known that caused by mutation is accidentalia, mutational site is at what
What kind of mutation place has carried out, and is unpredictable, it is new whether mutation produces later for one of ordinary skill in the art
Function, if influence the function of gene, be equally unpredictable, especially for the base on Chinese cabbage A01 chromosomes
For Bra013400, due to still belonging to technical blank to its research in terms of Chinese-cabbage Germplasm identification at present, because
This, present invention discover that Bra013400 genes sudden change region and the sudden change region function have breakthrough meaning.
The present invention is directed to the sudden change region of above-mentioned discovery, according to its both sides conserved regions design special primer, and for primer
Design be also not that conventional primer design method institute is getable, because of " GAGTTA " mutation of the sudden change region of the present invention
For " G ", that is, five bases of AGTTA are lacked, which is located at the code area of gene Bra013400 at initiation codon, makes
At frameshift mutation, therefore, how by design of primers, it is difficult point place to make amplified fragments include above-mentioned saltation zone；Moreover, this hair
How bright sudden change region only than wild type few five bases efficiently separate the segment realization after amplification and identification are this hairs
Where bright another difficult point.Based on above-mentioned factor, the present invention in design primer special consideration should be given to annealing temperature between 60-65 DEG C,
G/C content is no less than 40%, does not have mispairing, and amplified fragments are between 80-200bp, in combination with 8% non-denaturing polyacrylamide
Gel is separated by electrophoresis, and the identification to two kinds of genotype of Bra013400 genes wild type and saltant type is effectively realized.
In order to enable those skilled in the art can clearly understand technical scheme of the present invention, below with reference to tool
The embodiment of the body technical solution that the present invention will be described in detail.
Embodiment 1：Two kinds of typical case Chinese cabbage inbred lines material 06-247 and gram for being preced with Bra013400 target sequences in 291
It is grand
1.1 Chinese cabbage extracting genome DNAs
(1) Seedling of Chinese Cabbage blade is put into the mortar of Liquid nitrogen precooler, powder is fully ground into liquid nitrogen；
(2) it waits for that liquid nitrogen volatilization is dry, is immediately transferred in the centrifuge tube of 2ml, 0.6ml, which is about added, per 100mg materials is preheated to
65 DEG C of CTAB extracting solutions after thawing, shake vigorously and mix well sample, 65 DEG C of water-baths, which are placed 40-60 minutes, makes cell cracking；
(3) after cracking, taking out sample makes it be completely cooled down to room temperature.Isometric chloroform is added
(chloroform), gently overturning makes mixing, is placed at room temperature for 10 minutes；
(4) room temperature, 12000rpm are centrifuged 15 minutes；
(5) upper strata aqueous phase is carefully sucked out with pipettor, is added in the centrifuge tube of new 1.5ml, the isopropanol of 500 μ l is added
(1:1 volume), it mixes well, precipitation at room temperature 10min；
(6) 4 DEG C, 12000rpm centrifuges 10min, carefully discards supernatant liquid；
(7) DNA precipitations are washed with 75% ethyl alcohol of 1ml.4 DEG C, 8000rpm centrifuges 10min and collects precipitation；
(8) it repeats to washed once DNA precipitations with 75% ethyl alcohol；
(9) supernatant is removed, DNA, which is deposited on aseptic operating platform, to be dried about 10-15 minutes, and DNA shows slightly transparent, and appropriate bulk is added
The Tris.HCl (pH8.0) of the 10mM of product (30-50 μ l) makes precipitation dissolving (can be placed on 4 DEG C of refrigerator dissolvings overnight)；
(10) ultraviolet specrophotometer and 1%Agrose detected through gel electrophoresis DNA concentration and quality.
The clone of 1.2 Bra013400 target sequences and detection
(1) information provided according to sequencing file finds that variable region is located at the 5447932 of A01 chromosome physical locations
Place, " GAGTTA " herein sport " G ", that is, lack five bases of AGTTA, which is located at the coding of gene Bra013400
Frameshift mutation is caused at initiation codon in area.From in database copy Chinese cabbage A01 chromosomes on the both sides 5447932bp
The conserved sequence of each 100bp, in variant sites both sides conserved regions design primer, the design of primer special consideration should be given to annealing temperature between
60-65 DEG C, G/C content be no less than 40%, there is no mispairing, because insertion and deletion only has 5 bases, polyacrylamide electricity can only be passed through
Swimming is detected differentiation, so special consideration should be given to the big palpulus of amplified fragments between 80-200bp.By screening, upstream and downstream is finally determined
Primer sequence is as follows：Bra013400F：5'AAGAAATCTTCGAGTCGGAGGG-3' and Bra013400R：5'
AGACGGAAACTAGTCCTATCGGGTA-3', as shown in Seq ID No.3 and Seq ID No.4.
(2) PCR amplification：In 20 μ l reaction systems, including 1 × PCR masterMIX (containing dyestuff), the genome of 20ng
DNA；0.4 μM positive/negative to primer.PCR cycle condition：95 DEG C of pre-degenerations 5 minutes；It is denaturalized 30 seconds followed by 95 DEG C, 62 DEG C of annealing
30 seconds, 72 DEG C extended 10 seconds, and 35-38 cycle, last 72 DEG C extend 5 minutes.
(3) electrophoresis detection of PCR product：
After PCR, 1 μ l pcr amplification products is taken to carry out electrophoresis, electrophoresis on 8% non-denaturing polyacrylamide gel
After cma staining, digital camera takes pictures.As a result see Fig. 2.The segment amplified from two parts of materials is between 100bp-
Between 150bp, the amplified production in two materials is had any different in clip size, may be used as molecular labeling.
(4) sequence verification of PCR product
After electrophoresis confirms that purpose band is amplified, then with high fidelity enzyme purpose section is expanded, takes 1 μ l PCR amplifications
Product adds 1 μ l pEasy-Blunt carrier room temperatures to connect 10 minutes, conversion competent escherichia coli cell Trans1-T1
(TransGen：CD501), transformed bacteria is inverted 16 hours left sides of culture for 37 DEG C on the LB solid plates containing 50 μ g/ml kanamycins
It is right.Picking positive colony entrusts the measurement of Beijing Hua Da gene Co., Ltd progress DNA sequence dna after bacterium colony PCR detections.To gained
Sequence is compared, as a result consistent as shown in Figure 3 with weight sequencing data, by being compared with Bra013400 full genomes code area, sends out
Existing absent region is located at the ends 5'- of gene at initiation codon, and frameshift mutation causes the codon of mutator cannot be just
It often reads and terminates (as shown in Figure 4) in advance,.
Since primer combination Bra013400F/Bra013400R can be amplified in being selfed based material 2011-243
The band of 126bp can expand the band of 121bp in hat 291, and can be carried out by native polyacrylamide gel electrophoresis
It detecting (as shown in Figure 3), then this can be used to distinguish whether Bra013400 genes to be mutated in detection zone to primer,
Amplified production in 06-247 be exactly with the directly related label of detection wild type gene, be named as Bra013400W, sequence
Such as Seq ID No.1, the amplified production in hat 291 is exactly to detect directly related label with mutator, is named as
Bra013400m, sequence is as shown in Seq ID No.2.
The application that embodiment 2 marks
For the practicability of verification mark, we have detected the application with the combination of Bra013400F/Bra013400R primers
The part germ plasm resource of inventor's initiative, while F2 groups are constructed for parent with Chinese cabbage inbred lines 06-247 and hat 291, it uses
The primer combines the part single plant for having detected F2 groups, further demonstrates the practicability of label.It is as follows that detailed rules and regulations are embodied：
(1) each single plant extracting genome DNA of part germ plasm resource and F2 groups is as described in " 1.1 " in embodiment 1.
(2) PCR amplification：The preparation of PCR reaction solution and amplification condition are as described in " 1.2 " (2) article in embodiment 1.
(3) detection of PCR product is as described in " 1.2 " (3) article in embodiment.Testing result is as shown in figure 5, A is part
The testing result of germ plasm resource, wherein CK1 are that self-mating system 06-247, CK2 is that be preced with 291,1-10 be ten portions of Chinese cabbages money to self-mating system
Source, wherein the same CK1 of 1,5,8,10 genotype in the sites Bra013400, is wild type, remaining same CK2, gene has occurred prominent
Become；Fig. 5 B are the testing results for deriving F2 population segment single plants with hat 291 to 06-247, and P1 is that 06-247, P2 are hats 291,11-
20 be 10 single plants in F2 groups, as can be seen from the figure genotype same P1 of the number 15,18 in the sites Bra013400, is
Wild type, number 11,13,14,17,19 are mutant with parent P2, and 12,16,20 be heterozygous.It can be seen that label is to big
In the qualification result rate of accuracy reached 100% in the site, which can be in breeding practice for Chinese cabbage resource and breeding introgressive line
Early stage be detected, working efficiency can be significantly improved.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
SEQUENCE LISTING
<110>Vegetable or flower research institute of Shandong Academy of Agricultural Sciences
<120>The InDel of Chinese cabbage Bra013400 frameshift mutations is marked and its application in the practices of breeding
<130> 2017
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 140
<212> DNA
<213>Chinese cabbage
<400> 1
aagaaatctt cgagtcggag ggggataagc cgccgccggc tagaacggtg ttctagttcc 60
ttcgctgaag atctctcagc ctcgacttct tctttcgcga cgtgctcgat ggagttaccc 120
gataggacta gtttccgtct 140
<210> 2
<211> 135
<212> DNA
<213>Chinese cabbage
<400> 2
aagaaatctt cgagtcggag ggggataagc cgccgccggc tagaacggtg ttctagttcc 60
ttcgctgaag atctctcagc ctcgacttct tctttcgcga cgtgctcgat ggcccgatag 120
gactagtttc cgtct 135
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence
<400> 3
aagaaatctt cgagtcggag gg 22
<210> 4
<211> 25
<212> DNA
<213>Artificial sequence
<400> 4
agacggaaac tagtcctatc gggta 25
Claims (10)
1. a kind of codominant marker of the encoding gene Bra013400 wild types and mutant of identification Chinese cabbage TNL-WRKY,
It is characterized in that, the codominant marker includes：With the relevant label Bra013400W of wild type, clip size 140bp, such as SEQ
Shown in ID No.1；With mutant relevant label Bra013400m, size 135bp, as shown in SEQ ID No.2.
2. application of the codominant marker described in claim 1 in Chinese-cabbage Germplasm identification and/or breeding for disease resistance.
3. the primer sets for differentiating codominant marker described in claim 1, which is characterized in that the sequence of the primer sets is such as
Shown in SEQ ID No.3 and SEQ ID No.4.
4. the primer sets described in claim 3 are being prepared for identifying that the encoding gene Bra013400 of Chinese cabbage TNL-WRKY is wild
Application in the kit of raw type and mutant.
5. a kind of kit for identifying the encoding gene Bra013400 wild types and mutant of Chinese cabbage TNL-WRKY,
It is characterized in that, the kit includes：Primer shown in SEQ ID No.3 and SEQ ID No.4.
6. the kit described in primer sets or claim 5 described in claim 3 is identified and/or auxiliary in Chinese-cabbage Germplasm
Help the application in breeding.
7. application as claimed in claim 6, which is characterized in that application process is：Using described in claim 3 primer sets or
Kit described in claim 5 carries out PCR amplification to the genomic DNA of test individual；According to the size of amplified fragments to base
Because the TNL-WRKY encoding gene Bra013400 types contained in group are judged.
8. the use as claimed in claim 7, which is characterized in that judgment method is：If amplifying the band of 140bp, base
Because containing wild type gene in group；If amplifying the band of 135bp, which is mutated.
9. a kind of method that identification or auxiliary identify Chinese cabbage cultivar to be measured, which is characterized in that include the following steps：
(1) PCR amplification is carried out to the genomic DNA of test individual using the primer sets described in claim 3；
(2) size of detection amplified fragments contains wild type gene if amplifying the band of 140bp in genome；If
The band of 135bp is amplified, then the gene is mutated.
10. method as claimed in claim 9, which is characterized in that in step (1), the condition of PCR amplification is：95 DEG C of pre-degenerations 5
Minute；95 DEG C are denaturalized 30 seconds, and 62 DEG C are annealed 30 seconds, and 72 DEG C extend 10 seconds, 35-38 cycle；Last 72 DEG C extend 5 minutes.
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Cited By (1)
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|---|---|---|---|---|
| WO2020249111A1 (en) * | 2019-06-14 | 2020-12-17 | 山东大学 | Method and kit for detecting genome editing and application thereof |
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| US20110179521A1 (en) * | 2008-07-31 | 2011-07-21 | Riken | Plant showing resistance to multiple diseases and method for producing the same |
| EP2356899A1 (en) * | 2008-07-31 | 2011-08-17 | Riken | Plant having resistance to multiple diseases and method for production thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020249111A1 (en) * | 2019-06-14 | 2020-12-17 | 山东大学 | Method and kit for detecting genome editing and application thereof |
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