CN107937599A - A kind of molecular labeling KB2, primer and application for identifying the anti-clubroot of Chinese cabbage - Google Patents
A kind of molecular labeling KB2, primer and application for identifying the anti-clubroot of Chinese cabbage Download PDFInfo
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Abstract
The invention discloses a kind of and celery cabbage clubroot resistant geneCrr1aInDel molecular labelings KB2, primer and its application isolated.The present invention provides identify or assisting in differentiate whether Chinese cabbage contains anti-knee ospc geneCrr1a, if it is the method for anti-clubroot Chinese cabbage, including using Chinese cabbage genome to be detected as template, PCR amplification is carried out with primer provided by the invention, according to the size of amplified production, judges whether Chinese cabbage to be detected contains anti-knee ospc geneCrr1a, if it is anti-clubroot Chinese cabbage.Assisted Selection is carried out using the molecular labeling confrontation clubroot Chinese cabbage of the present invention, accuracy is high, and cost is low, time saving and energy saving.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of molecular labeling KB2 for differentiating celery cabbage clubroot resistance,
Primer and its application.
Background technology
Chinese cabbage（Brassica rapa L. ssp. pekinensis）Belong to Cruciferae Brassica genus, be China's plantation
One of vegetable crop of area and yield maximum.Clubroot is by plasmodiophora brassicae（Plasmodiophora brassicae
Woronin）Caused soil Hereditary diseases, can cause the root exception enlargement of the crucifers such as Chinese cabbage, form root nodule, be
A kind of serious global crucifer root disease, can cause subtracting for global brassicaceous vegetable 10%-15% every year
Production, in the serious area of morbidity, or even causes to have no harvest, the production of serious threat brassicaceous vegetable.The disease is found in Europe earliest,
In recent years, overwhelming majority provinces, cities and autonomous regions in China are all related to the pathogenetic report of knee, particularly Yunnan, Hubei, Liaoning,
The areas such as Shandong, the production to vegetables such as China Chinese cabbages cause tremendous influence.
Although physics and chemistry etc. can be taken to prevent plasmodiophora brassicae, cost is higher, and effect and unobvious.Training
The new varieties of anti-clubroot are educated, are that prevention celery cabbage clubroot is most economical, most effective measure.Traditional anti-clubroot breeding
In, per a generation be required for using Field inoculation pathogen carry out Characters Identification method, it is time-consuming and laborious, and easily be subject to environment because
The influence of element.Molecular marker assisted selection（Marker-assisted selection, MAS）Technology provides for breeding work
Tool, the technology are to utilize the molecular labeling chain with target gene, and all objective traits are completed by the detection of DNA level
Selection, improve efficiency of selection, reduce workload, accelerate breeding process.
Using Williams identification systems, the biological strain of plasmodiophora brassicae is identified, it is found that No. 4 biological strains are China
The sociales of plasmodiophora brassicae, pathogenic strong, harm is the most serious.In Chinese cabbage different chromosomes, at least find that 8 clubroots resist
Property gene or QTLs, wherein, on Chinese cabbage A08 chromosomes, there is an anti-knee ospc geneCrr1a, have to No. 4 biological strains
It is resistant.Although there is part to be announced with the chain molecular labeling in the site, its linksystem is not high enough, there is certain false positive
Produce, or during detection, disease-resistant and susceptible materials variances unobvious, as a result easily obscure.Suitable molecular labeling is developed, is used for
The selection and breeding of the anti-clubroot kind of Chinese cabbage or strain, it is possible to reduce workload, improves work efficiency, reduces breeding cost, accelerates
Breeding process, is of very high actual application value.
The content of the invention
Selection and breeding for the anti-clubroot kind of Chinese cabbage or strain, conventional method are needed often for all in field progress plasmodiophora brassicae
Inoculated identification disease resistance, has the shortcomings that time-consuming, laborious easily affected by environment, and the present invention provides one kind to identify Chinese cabbage knee
InDel molecular labelings, primer and its application of sick resistance, DNA level can be carried out using the molecular labeling in Chinese Cabbage
Identification so that Chinese cabbage overcome needed in traditional breeding method be inoculated with pathogen identification disease resistance the shortcomings that, simplify anti-of Chinese cabbage
The cultivating process of swollen disease kind or strain.
The present invention provides it is a kind of differentiate or auxiliary differentiate Chinese cabbage whether be anti-clubroot Chinese cabbage InDel molecules
KB2 is marked, the molecular labeling single-stranded DNA sequence is from 5 ' ends to 3 ' ends：
CTCAAGCTGTGCTCAAGCCTTATGGAACTCCCCTCTTCTATTGGTAACATGACTAATCTTGAGAATTTGAATCTTTC
TGGATGCTCTAGCCTTGTGGAGCTCCCCTCTTCTATTAGTAATATGACTAATCTTGAGAATTTTAATCTCTCCCAAT
GCTCAAGCGTTGTACGGCTCTCTTTTTCTATTGGAAATATGACCAATCTCAAG
Above-mentioned identify whether Chinese cabbage is that the specificity of molecular labeling of anti-clubroot Chinese cabbage is drawn present invention also offers a kind of
Thing, its single-stranded DNA sequence are from 5 ' ends to 3 ' ends：
Sense primer F: CTCAAGCTGTGCTCAAGCCTTATGG
Anti-sense primer R: ATTGAGATTGGTCATATTTCCAATA
The present invention can be achieved through the following technical solutions：The genomic DNA of cabbage samples to be detected is extracted as template, is utilized
The present invention molecular labeling specific primer carry out PCR (PCR) amplification, then by electrophoresis to amplified production into
Row detection.
Comprise the following steps that：
1) the cabbage samples to be measured arbitrarily genomic DNA of tissue or organ is extracted using CTAB methods；
2) using the sample to be tested genomic DNA obtained by step 1 as template, using molecular labeling specific primer to different materials
PCR amplification is carried out, obtains amplified production；
3) agarose gel electrophoresis technology is utilized, the pcr amplification product obtained by step 2 is separated by electrophoresis；
4) gel imaging system is utilized, observes the position of pcr amplification product；
5) by judging the size of amplified production, the larger DNA fragmentation for 207bp of amplified production, expands containing 207bp and produces
The sample to be tested of thing contains anti-knee ospc geneCrr1a, swell disease Chinese cabbage material for anti-liver；207bp amplified productions are not contained
Sample to be tested does not contain anti-knee ospc geneCrr1a, it is non-anti- clubroot Chinese cabbage material.
Molecular labeling specific primer used in above-mentioned PCR amplification is as follows（1）With（2）：
（1）As the single strand dna shown in sequence in sequence table 2, or sequence 2 deleted, increased or be mutated is one or several
Nucleotide, and with sequence 2 have identical function single strand dna.
（2）As the single strand dna shown in sequence in sequence table 3, or by sequence 3 delete, increase or be mutated one or
Several nucleotide, and with sequence 3 have identical function single strand dna.
A kind of and celery cabbage clubroot resistant gene disclosed by the inventionCrr1aThe InDel molecular labelings KB2 that isolates,
Primer compared with prior art, the beneficial effects of the invention are as follows：
（1）By being detected to the direct agarose electrophoresis of pcr amplification product, can be clearly distinguished whether Chinese cabbage material is anti-
Swollen disease Chinese cabbage material, it is time saving and energy saving, from such environmental effects.
（2）The molecular labeling and its specific primer that the present invention obtains, can be right in different Chinese cabbage muskmelon materials
Anti- clubroot Chinese cabbage is distinguished well, therefore, present invention obtains one can efficiently, whether accurately distinguish Chinese cabbage
For the molecular labeling of anti-clubroot Chinese cabbage material.
（3）The method of the present invention specificity is good, and accuracy rate can reach 100%.
（4）The present invention is easier than polyacrylamide gel electrophoresis detection fast by being detected to the direct agarose electrophoresis of PCR product
It is prompt；Detection than SNP marker reduces cost, is the very strong molecular labeling of practicality.
The present invention of table 1 is compared with prior art advantage and disadvantage
Brief description of the drawings
Fig. 1 is the disease-resistant material of clubroot and the susceptible phenotype of susceptible material field clubroot：Wherein G4 represents that clubroot is susceptible
Material, G6 represent the disease-resistant material of clubroot；
Fig. 2 is the specific primer of molecular labeling of the present invention with anti-clubroot material G6, sense clubroot material G4 and F2 generation separation
The genomic DNA of colony carries out the electrophoresis result of PCR amplification for template：Wherein M represents DNA Marker, remaining swimming lane represents not
It it is respectively No. 1 with the electrophoresis result of the pcr amplification product of cabbage samples：The susceptible material G4 of clubroot；No. 2：Anti- clubroot material
Expect G6, remaining sample is F2 for segregating population material.
Embodiment：
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention is
Method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, to be not intended to limit the present invention model
Enclose, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this hair
On the premise of bright spirit and scope, the various changes or change that are carried out to the material component in these embodiments and dosage also belong to
In protection scope of the present invention.Experimental method used in following embodiments, is conventional method unless otherwise specified；Institute
Instrument, material and reagent etc., unless otherwise specified, commercially obtain.
The susceptible material G4 of Chinese cabbage material clubroot, anti-clubroot material G6 and F1 generation material in following embodiments, it is public
Crowd can obtain from Chinese cabbage seminar of Inst. of Vegetable, Kerun Agriculture Sci-Tech Co Ltd, Tianjin, which only attaches most importance to duplicate
Used in the related experiment of invention, it can not be used as other purposes.
Embodiment 1
The acquisition of the disease-resistant material F2 segregating populations of celery cabbage clubroot
Using the anti-clubroot material G6 of Chinese cabbage as female parent, susceptible material G4 is male parent, carries out hybridization and obtains F1 generation, F1 generation plant warp
Field resistance identification, all disease-resistant materials.F1 generation selfing obtains F2 for segregating population, reflects for celery cabbage clubroot phenotype
Fixed and Markers for Detection.
Embodiment 2.
Chinese cabbage F2 is for colony's clubroot phenotypic evaluation and Markers for Detection
First, F2 colonies celery cabbage clubroot resistant phenotype is identified
The F2 colonies obtained using embodiment 1 are after planting inoculated with knee germina number-four biological strain, inoculation and investigation method ginseng as material
According to document " Wang Weihong etc., Hubei governor's sun county Plasmodiophora brassicae Causing Cruciferae Clubroot Race Identification and resistance screening, Chinese vegetable
Dish, 2013 Vol. 1 (12)：Method in 55-60 " carries out.The result shows that：For in Chinese cabbage plant, 47 plants show 64 plants of F2
To be disease-resistant, 17 plants show as susceptible (Fig. 1).
2nd, the Markers for Detection of F2 colonies celery cabbage clubroot resistance
Using CTAB methods extraction clubroot resistant material G6, susceptible material G4, F2 for segregating population material blade genome
DNA, specific step are：Take 0.2 g of young leaflet tablet to enter in 1.5 mL centrifuge tubes, add 50 μ L, 2% CTAB Extraction buffers, grind
Mill, rear polishing to 400 μ L, 65 DEG C of 30 min of water-bath；Add 400 μ L chloroforms：Isoamyl alcohol（24:1）, 5 min of jog.12000
Rpm centrifuges 5 min；200 μ L of supernatant are taken, the 200 μ L of isopropanol for adding precooling are mixed, -20 DEG C of 20 min of placement；12000
Rpm centrifuges 10 min；Supernatant is abandoned, adds 150 μ L pre-cooled ethanols, gently mixes and cleans, 10,000 rpm centrifuge 5 min；Abandon
Clearly, dry or dry up；Add 100 μ L dissolving DNAs of distilled water, room temperature places 1 h；DNA is diluted to 50 ng/ μ L with distilled water,
Used as pcr template or -20 DEG C save backup.
Using above-mentioned genomic DNA as template, molecular labeling specific primer carries out PCR amplification, obtains amplified production, specifically
Property primer is made of two single stranded DNAs of sense primer F and anti-sense primer R, its sequence is as follows：
Sense primer F: CTCAAGCTGTGCTCAAGCCTTATGG
Anti-sense primer R: ATTGAGATTGGTCATATTTCCAATA
Using the reaction system of 10 μ L, including：10 × PCR Buffer, 1.0 μ L；2.5 mM dNTP, 0.2 μ L；10 µ
mol·L-1Primers, 0.4 μ L；5 U·µL-1Taq, 0.2 μ L；50 ng·µL-1DNA, 1.5 μ L；ddH2O, 6.7 μ
L。
The program that PCR amplification uses for：94 DEG C of 5 min of pre-degeneration；94 DEG C of 30 s of denaturation, 57 DEG C of 30 s of renaturation, 72 DEG C are prolonged
Stretch 30 s, 35 circulations；72 DEG C extend 5 min eventually.
To pcr amplification product in 2% Ago-Gel, after 150 v constant pressures electrophoresis 30 min, EB dyeing, with gel into
As system observation result.Kit can also be made in specific primer and PCR other compositions, for identify Chinese cabbage whether be
Anti- clubroot Chinese cabbage.Wherein, the band that a size is 207bp is only amplified in disease-resistant parent G6, in Susceptible parent G4 only
The band of a 136bp is amplified, F1 generation amplifies two bands of 207bp and 136bp at the same time.The result shows that in F2 for group
In body, clubroot resistant phenotype is accredited as 17 plants of susceptible materials, and all only amplification obtains the band of 131bp, with Susceptible parent
Unanimously；Phenotypic evaluation is 47 plants of disease-resistant materials, and amplification obtains the band of 207bp, wherein 19 plants of materials are only expanded to 207bp
Band, the amplification of 28 plants of materials arrives two bands containing 207bp and 136bp（Fig. 2）.Molecular labeling is to the anti-clubroot of Chinese cabbage
Testing result and phenotypic evaluation result are completely the same.
The band for resisting 207bp in the pcr amplification product of the Chinese cabbage plant of clubroot is sequenced, the results showed that：
Single stranded nucleotide sequence of the sequence of the DNA fragmentation of 207bp as shown in sequence 1 in sequence table, i.e., molecular labeling in the application
Sequence.It follows that the result of molecular labeling KB2 and its primer pair celery cabbage clubroot Molecular Detection is reflected with phenotype in the application
Fixed result is completely the same, the clubroot resistant gene of nucleotide sequence and Chinese cabbage shown in molecular labeling KB2Crr1aIt is
Mark is isolated, can be identified or aided in identify using the clubroot resistance of the sequence and its primer pair Chinese cabbage.
SEQUENCE LISTING
<110>Tianjin Kerun Agricultural Science ＆ Technology Co., Ltd.
<120>A kind of molecular labeling KB2, primer and application for identifying the anti-clubroot of Chinese cabbage
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 207
<212> DNA
<213>Artificial sequence
<400> 1
ctcaagctgt gctcaagcct tatggaactc ccctcttcta ttggtaacat gactaatctt 60
gagaatttga atctttctgg atgctctagc cttgtggagc tcccctcttc tattagtaat 120
atgactaatc ttgagaattt taatctctcc caatgctcaa gcgttgtacg gctctctttt 180
tctattggaa atatgaccaa tctcaag 207
<210> 2
<211> 25
<212> DNA
<213>Artificial sequence
<400> 2
ctcaagctgt gctcaagcct tatgg 25
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
attgagattg gtcatatttc caata 25
Claims (8)
1. it is a kind of be used for differentiate or aid in differentiate Chinese cabbage whether be anti-clubroot Chinese cabbage InDel molecular labeling KB2, its
It is characterized in that, single-stranded DNA sequence of the molecular labeling from 5 ' ends to 3 ' ends is：
CTCAAGCTGTGCTCAAGCCTTATGGAACTCCCCTCTTCTATTGGTAACATGACTAATCTTGAGAATTTGAATC
TTTCTGGATGCTCTAGCCTTGTGGAGCTCCCCTCTTCTATTAGTAATATGACTAATCTTGAGAATTTTAATCTCTCC
CAATGCTCAAGCGTTGTACGGCTCTCTTTTTCTATTGGAAATATGACCAATCTCAAG。
2. it is a kind of for described in claim 1 identify Chinese cabbage whether be anti-clubroot Chinese cabbage molecular labeling specificity
Primer, the single-stranded DNA sequence at its 5 ' end to 3 ' ends are：
Sense primer F: CTCAAGCTGTGCTCAAGCCTTATGG
Anti-sense primer R: ATTGAGATTGGTCATATTTCCAATA.
3. using the method for the InDel molecular labelings KB2 identification celery cabbage clubroots described in claim 1, it is characterized in that extraction
The genomic DNA of cabbage samples to be detected as template, using the molecular labeling specific primer described in claim 2 into
Row PCR amplification, then amplified production is detected by electrophoresis.
4. identifying whether Chinese cabbage is the method for anti-clubroot Chinese cabbage described in claim 3, including identify Chinese cabbage gene
Group DNA in whether the DNA fragmentation containing the 207bp shown in claim 1, if in the genome of cabbage samples to be detected
DNA fragmentation containing the 207bp shown in claim 1, Chinese cabbage to be detected contain anti-knee ospc geneCrr1a, for anti-
Swollen disease Chinese cabbage or the anti-clubroot Chinese cabbage of candidate；If claim is not contained in the genome of cabbage samples to be detected
The DNA fragmentation of 207bp shown in 1, Chinese cabbage to be detected do not contain anti-knee ospc geneCrr1a, it is non-anti- clubroot great Bai
The anti-clubroot Chinese cabbage of dish or non-candidate.
5. the InDel molecular labelings KB2 for being used to identify the anti-clubroot of Chinese cabbage the answering in following areas described in claim 1
With：
A, the application in Chinese cabbage breeding；
B, the application in the anti-clubroot Chinese cabbage strain of selection and breeding or kind；
C, identifying or aiding in identify Chinese cabbage for the application in anti-clubroot breeds of Chinese cabbage or strain；
D, the application in prediction Chinese cabbage is anti-clubroot material.
6. the molecular labeling specific primer for being used to identify the anti-clubroot of Chinese cabbage described in claim 2 is in following areas
Using：
A, the application in Chinese cabbage breeding；
B, the application in the anti-clubroot Chinese cabbage strain of selection and breeding or kind；
C, identifying or aiding in identify Chinese cabbage for the application in anti-clubroot breeds of Chinese cabbage or strain；
D, the application in prediction Chinese cabbage is anti-clubroot material.
7. it is a kind of containing described in claim 4 identify Chinese cabbage whether be anti-clubroot Chinese cabbage molecular labeling specificity
The kit of primer.
8. application of the kit in identifying whether Chinese cabbage is anti-clubroot Chinese cabbage described in claim 7.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588264A (en) * | 2018-06-08 | 2018-09-28 | 西南大学 | Molecular labeling, primer and its application for identifying club-root resistance |
| CN108866235A (en) * | 2018-08-29 | 2018-11-23 | 北京市农林科学院 | A kind of InDel molecular labeling and its application for identifying or assisting to identify Chinese cabbage crossing compatibility |
| CN110734999A (en) * | 2019-11-12 | 2020-01-31 | 北京市农林科学院 | SNP molecular marker tightly linked with new clubroot-resistant gene CRw of Chinese cabbage and application thereof |
| CN111748643A (en) * | 2020-07-03 | 2020-10-09 | 河南省农业科学院园艺研究所 | SNP molecular marker of gene CRs related to Chinese cabbage clubroot disease resistance and application thereof |
| CN112143823A (en) * | 2020-05-15 | 2020-12-29 | 河南省农业科学院园艺研究所 | KASP marker of Chinese cabbage clubroot resistance gene Crr5 and application thereof |
| CN112553359A (en) * | 2021-02-04 | 2021-03-26 | 沈阳农业大学 | Clubroot molecular marker syau3008 co-separated from Chinese cabbage gene, primer and application |
| CN112831594A (en) * | 2021-03-17 | 2021-05-25 | 西北农林科技大学 | Functional molecular marker of clubroot-resistant gene CRa of Chinese cabbage and application of functional molecular marker |
| CN113201596A (en) * | 2021-01-13 | 2021-08-03 | 湖北省农业科学院经济作物研究所 | InDel molecular marker primer for identifying clubroot-resistant gene CRb of cruciferous plant and application |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588264A (en) * | 2018-06-08 | 2018-09-28 | 西南大学 | Molecular labeling, primer and its application for identifying club-root resistance |
| CN108588264B (en) * | 2018-06-08 | 2021-05-28 | 西南大学 | Molecular marker and primer for identifying cabbage clubroot disease resistance and application of molecular marker and primer |
| CN108866235A (en) * | 2018-08-29 | 2018-11-23 | 北京市农林科学院 | A kind of InDel molecular labeling and its application for identifying or assisting to identify Chinese cabbage crossing compatibility |
| CN108866235B (en) * | 2018-08-29 | 2022-03-22 | 北京市农林科学院 | InDel molecular marker for identifying or assisting in identifying cabbage hybridization affinity and application thereof |
| CN110734999A (en) * | 2019-11-12 | 2020-01-31 | 北京市农林科学院 | SNP molecular marker tightly linked with new clubroot-resistant gene CRw of Chinese cabbage and application thereof |
| CN110734999B (en) * | 2019-11-12 | 2020-06-16 | 北京市农林科学院 | SNP molecular marker tightly linked with new clubroot-resistant gene CRw of Chinese cabbage and application thereof |
| CN112143823A (en) * | 2020-05-15 | 2020-12-29 | 河南省农业科学院园艺研究所 | KASP marker of Chinese cabbage clubroot resistance gene Crr5 and application thereof |
| CN112143823B (en) * | 2020-05-15 | 2022-08-16 | 河南省农业科学院园艺研究所 | KASP marker of Chinese cabbage clubroot resistance gene Crr5 and application thereof |
| CN111748643A (en) * | 2020-07-03 | 2020-10-09 | 河南省农业科学院园艺研究所 | SNP molecular marker of gene CRs related to Chinese cabbage clubroot disease resistance and application thereof |
| CN113201596A (en) * | 2021-01-13 | 2021-08-03 | 湖北省农业科学院经济作物研究所 | InDel molecular marker primer for identifying clubroot-resistant gene CRb of cruciferous plant and application |
| CN113201596B (en) * | 2021-01-13 | 2022-09-16 | 湖北省农业科学院经济作物研究所 | InDel molecular marker primer for identifying clubroot-resistant gene CRb of cruciferous plant and application |
| CN112553359A (en) * | 2021-02-04 | 2021-03-26 | 沈阳农业大学 | Clubroot molecular marker syau3008 co-separated from Chinese cabbage gene, primer and application |
| CN112553359B (en) * | 2021-02-04 | 2023-05-05 | 沈阳农业大学 | Clubroot molecular marker syau3008 coseparated from Chinese cabbage genes, primer and application |
| CN112831594A (en) * | 2021-03-17 | 2021-05-25 | 西北农林科技大学 | Functional molecular marker of clubroot-resistant gene CRa of Chinese cabbage and application of functional molecular marker |
| CN112831594B (en) * | 2021-03-17 | 2023-03-14 | 西北农林科技大学 | Functional molecular marker of clubroot-resistant gene CRa of Chinese cabbage and application of functional molecular marker |
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