CN105695609B - For the PCR primer of Cabbage Wilt Disease resistance screening, kit and its application - Google Patents
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Abstract
The invention discloses a kind of for the PCR primer of Cabbage Wilt Disease resistance screening, kit and its application, is related to molecular breeding technology.PCR primer for Cabbage Wilt Disease resistance screening is Frg13-F/Frg13-R, and nucleotide sequence is as shown in Seq ID No.1 and Seq ID No.2.There is the wild cabbage of fusarium wilt disease resistance using primer provided by the invention or kit assisting in test of wilt disease resistance in brassica oleracea and/or assisting sifting, coincide rate up to 96% with field test result.PCR primer of the invention, kit or method, which are used for breeding, to be had many advantages, such as that easy to operation, high specificity, stability are good, early stage breeding may be implemented, and has major application prospect.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of PCR of assisting in test of wilt disease resistance in brassica oleracea is dedicated to be drawn
Object, kit and its application.
Background technique
Wild cabbage (Brassica oleracea L.) class vegetables are Cruciferae (Cruciferae) Brassica genus
(Brassica) general designation of the biennial vegetable in brassica specie, wild cabbage adaptability and resistance are relatively strong, are many in the world
The main vegetables crop of country, in also cultivation extensively all over China.The main crops for rotation of China's wild cabbage have spring wild cabbage, summer wild cabbage, autumn
Wild cabbage and extremely frigid zones 1 year one season planting type, at the beginning of 21 century, China's wild cabbage cultivated area has reached 900,000 hectares.
Cabbage Wilt Disease is found in the U.S. earliest, is found at present in the whole world most of summer and autumn wild cabbage cultivations area.
In China, Cabbage Wilt Disease first discovery has expanded to the most laboratories in wild cabbage producing region in Yanqing County of Beijing area in 2001.
The pathogenic bacteria of Cabbage Wilt Disease are the glutinous group's transformants of Fusarium oxysporum.Cabbage Wilt Disease mostly be during being colonized or loosening the soil,
The wound invaded plants that generate from tender supporting root or old root expand to xylem, then by stem up to blade,
Germ is in the organization internal infected and external generation spore, until part or whole strain are dead.The diseased plant endangered by Cabbage Wilt Disease
Its Symptoms are as follows: firstly, diseased plant lower blade turns yellow, the blade in addition to vein still maintains green；Then, upper blade by
Gradual change is yellow, is equally first to turn yellow in blades whole after netted yellow；Last whole blade flavescence is wilted, growing point and stem are downright bad,
Entire plant is wilted and gradually death, the shortening stem of diseased plant, petiole etc. is cut, it is possible to find vascular bundle part browning.Wild cabbage is withered
The occurrence and development for disease of withering are influenced by many factors such as the soil moisture, humidity, but temperature is primary factor, high temperature, drying
Soil environment is easier to fall ill.
Cabbage Wilt Disease is a kind of typical soil-borne disease, can be accumulated year by year in the continuously soil of plantation Brassica Crops
It is tired, so that harm gradually aggravates.And in the field that wilt disease occurred, even if no longer planting Brassica Crops, pathogen
It is more than energy Survival for 10 Years, therefore prevention and control of the means such as traditional agriculture means of prevention such as seed treatment, crop rotation, chemical control to the disease
Effect is unobvious, is the most effective approach for eliminating this destructive disease potential threat using resistant variety.
Although conventional disease resistant and breeding method has played significant role, but there is also many disadvantages.Conventional breeding for disease resistance master
It to be carried out by hybridization and inbreeding of more generation, the complex processes such as the selection of disease-resistant material and field test, the period is relatively long, and
Vulnerable to environmental influence, reliability is poor.
Therefore, this field needs to develop a kind of PCR primer and related kit for assisting sifting containing Cabbage Wilt Disease
The wild cabbage material of resistant gene.
Summary of the invention
According to the demand of this field, the present invention provides a kind of PCR primer for Cabbage Wilt Disease resistance screening,
High specificity, stability is good, can quickly, effectively, be flexibly utilized by either phase cabbage plant filter out it is anti-containing wilt disease
Property gene wild cabbage material, be used for breeding.
The claimed technical solution of the present invention is as follows:
For screening the PCR primer of wild cabbage (Brassica oleracea L.) fusarium wilt disease resistance material, nucleotides sequence
It is classified as:
Upstream primer Frg13-F:5`-ACCAGAGGCAGTTTTGGTTG-3 '
Downstream primer Frg13-R:5`-TCTTGCAACCCATGTCAAAA-3 '；
The Cabbage Wilt Disease resistant material characteristic bands size of the PCR primer amplification is 263bp, and nucleotide sequence is such as
Shown in Seq ID No.3.
For screening the kit of Cabbage Wilt Disease resistant material, which is characterized in that including PCR described in claim 1
Primer.
The kit further includes for reagent needed for carrying out PCR and/or electrophoresis.
Method for screening Cabbage Wilt Disease resistant material, which is characterized in that including using the PCR primer and/
Or the kit carries out following steps:
(1) PCR is carried out using genomic DNA of the PCR primer to wild cabbage material to be measured；
(2) PCR product described in detected through gel electrophoresis；
(3) it is filtered out from the electrophoresis detection result in the same size with the Cabbage Wilt Disease resistant material characteristic bands
Material；
The Cabbage Wilt Disease resistant material characteristic bands are 263bp, and nucleotide sequence is as shown in Seq ID No.3.
The PCR reaction system are as follows: 0.2 μ L/ μ L of templet gene group DNA includes Mg2+10 × PCR buffer buffering
0.1 μ L/ μ L, dNTPs 0.2mM, Taq archaeal dna polymerase 0.5U/ μ L of liquid, forward primer and 0.4 μM of reverse primer, remaining is double
Steam water.
The reaction condition of the PCR are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C extend
45s, 35 circulations；72 DEG C of extension 7min；4 DEG C of preservations.
The detected through gel electrophoresis refers to the polyacrylamide gel using 8%, is separated by electrophoresis 70 minutes in 160V invariable power,
Last silver staining colour developing.
The preparation method of the kit, which is characterized in that in the packing box for indicating Cabbage Wilt Disease resistance screening purposes
It is provided with the PCR primer.
It is also equipped in the packing box for indicating Cabbage Wilt Disease resistance screening purposes for carrying out PCR and/or electrophoresis institute
The reagent needed.
The present invention designs to obtain according to disease-resistant wild cabbage material 96-100 and susceptible wild cabbage material 01-20 weight sequencing result.It is first
The simple repeated sequence informative site in weight sequencing sequence first is searched for using SSRHunter1.3, then chooses informative site nucleosides
Sour duplicated gene sequence is that 2-6 sequences are target sequence, designs SSR primer using software Primer 6.0, therefrom screens
Obtain specific primer.
Technical term:
It resurveys sequence: the species of known group sequence being carried out with the gene order-checking of Different Individual, and right on this basis
Individual or group carry out difference analysis.
Simple repeated sequence informative site: refer in genome with a few nucleotide (majority is 2-4) for unit
The sequence up to tens nucleotide of multiple tandem sequence repeats composition, also known as simple sequence repeats (Simple Sequence
Repeats,SSR)。
The present invention carries out Cabbage Wilt Disease resistant material as molecular labeling using the above-mentioned specific primer screened
Assisted Selection provides new approach for breeding and varietal resistance identification.Molecular labeling can show directly in the form of DNA,
It can be detected in each tissue of organism, each stage of development, not by season and environmental restrictions, accuracy and reliability
It is high.
PCR primer high specificity provided by the present invention, stability is good, any one annealing temperature between 53-62 DEG C
Non-specific amplification is not generated under degree, can sufficiently accurately detect whether there is fusarium wilt disease resistance in cabbage plant to be measured.
Under normal circumstances, the resistance of different cultivars wild cabbage material needs just to can determine that by the method for inoculated identification, and uses this hair
Bright primers F rg13-F/Frg13-R carries out the assisting sifting of Cabbage Wilt Disease resistance, can quickly filter out in Seedling Stage
Target strain, and inoculated identification is observed as a result, the conditions such as environmental condition need to be will receive by cumbersome inoculated identification process
It influences, repeatability is poor, as a result unreliable.Therefore, the breeding assisted Selection of wild cabbage is carried out using PCR label of the invention, greatly
It shortens breeding cycle greatly, improve breeding efficiency.
The present invention also provides a kind of methods for Cabbage Wilt Disease resistance assisting sifting, and operation is simple for this method,
The genomic DNA that wild cabbage to be measured need to only be extracted carries out PCR reaction using primers F rg13-F/Frg13-R, passes through polyacrylamide
The length of detected through gel electrophoresis PCR product characteristic bands can judge whether there is fusarium wilt disease resistance in plant to be measured.
In conclusion the screening of Cabbage Wilt Disease resistance is carried out using PCR provided by the invention label and primer, operation letter
Single easy, high specificity, stability is good, can greatly shorten breeding cycle, improves breeding efficiency.
Detailed description of the invention
Amplification situation of Fig. 1 primer in disease-resistant wild cabbage material, wherein M:DNA ladder；1-16: wild cabbage is anti-withered
Disease selfing based material " ZLR1 ", " ZLR2 ", " rare ", " summer is strong ", " 96-100 ", " ZLR3 ", " ZLR4 ", " ZLR5 ", " ZLR6 ",
"ZLR7","ZLR8","ZLR9","ZLR10","ZLR11","ZLR12","ZLR13"；17-32: wild cabbage sense wilt disease self-mating system
Material " gold early raw ", " 21-3 ", " ZLS1 ", " ZLS2 ", " ZLS3 ", " ZLS4 ", " 01-20 ", " 87-534 ", " ZLS5 ",
“ZLS6”、“ZLS7”、“ZLS8”、“ZLS9”、“ZLS10”、“ZLS11”、“ZLS12”。
Amplification situation of Fig. 2 primer under different annealing temperature, wherein A:53 DEG C；B:56 DEG C；C:59 DEG C；D:62 DEG C.
Specific embodiment
Below in conjunction with specific implementation example, the present invention is further explained, it should be noted that example is only used for explaining this hair
It is bright and do not limit the scope of the invention.The not specified experiment reagent of the present invention is this field conventional reagent, or using this
Field conventional method is prepared and is obtained, commercially available, and specification is the pure grade in laboratory.
Biomaterial
In the wild cabbage material used in embodiment 3 and embodiment 4, " ZLR1 ", " ZLR2 ", " ZLR3 ", " ZLR4 ",
" ZLR5 ", " ZLR6 ", " ZLR7 ", " ZLR8 ", " ZLR9 ", " ZLR10 ", " ZLR11 ", " ZLR12 ", " ZLR13 " and " ZLS1 ",
“ZLS2”、“ZLS3”、“ZLS4”、“ZLS5”、“ZLS6”、“ZLS7”、“ZLS8”、“ZLS9”、“ZLS10”、“ZLS11”
" ZLS12 " total 25 materials have preservation for self-mating system breeding material, applicant laboratory for height used in this laboratory, can
It is provided to the public for verifying the present invention in 20 years from the applying date.Remaining biomaterial is existing known materials, can quotient
Purchase obtains.
Embodiment 1, PCR primer of the present invention
Primer development process: primer of the present invention is according to disease-resistant wild cabbage material 96-100 and susceptible wild cabbage material 01-
20 weight sequencing results design to obtain.The simple repeated sequence information bit in weight sequencing sequence is searched for using SSRHunter1.3 first
Point, then choosing the sequence that informative site nucleotide duplicated gene sequence is 2-6 is target sequence, utilizes software Primer
6.0 design SSR primers.The major parameter of primer is primer length 20-26bp；48 DEG C -65 DEG C of annealing temperature Tm value；(G+C) contain
Measure 30%-70%；Pcr amplification product length is greater than 150bp.And the grade of fit of primer indices is verified with Oligo 6.65,
It sees either with or without primer dimer, hairpin structure.It is screened out from it qualified primer sequence and by the outstanding auspicious biotechnology in Shanghai
It Co., Ltd and is screened at rear using high sense and highly resistance selfing based material, finally obtains primer of the present invention.
The present embodiment provides a kind of for screening the PCR primer of Cabbage Wilt Disease resistant material, has following nucleotide
Sequence:
Upstream primer Frg13-F:5`-ACCAGAGGCAGTTTTGGTTG-3 ' (Seq ID No.1)
Downstream primer Frg13-R:5`-TCTTGCAACCCATGTCAAAA-3 ' (Seq ID No.2),
Above-mentioned PCR primer can be artificial synthesized.
The PCR primer expands big with the molecular marker characteristic band of Cabbage Wilt Disease resistant gene FOC1 close linkage
Small is 263bp, and nucleotide sequence is as shown in Seq ID No.3.
Embodiment 2, kit of the present invention
The present embodiment provides a kind of for screening the kit of Cabbage Wilt Disease resistant material comprising described in embodiment 1
PCR primer.
Further, the kit further includes for reagent needed for carrying out PCR and/or electrophoresis.
The production method of mentioned reagent box: embodiment is provided in the packing box for indicating Cabbage Wilt Disease resistance screening purposes
PCR primer described in 1.
Further, be also equipped in the packing box for indicating Cabbage Wilt Disease resistance screening purposes for carry out PCR and/
Or reagent needed for electrophoresis.
The Accuracy Verification of embodiment 3, the PCR primer or kit screening Cabbage Wilt Disease resistant material
1, genomic DNA is extracted
It is mentioned with CTAB (cetyltriethylammonium bromide, Hexadecyl trimethyl ammonium Bromide) method
Take wild cabbage " ZLR1 ", " ZLR2 ", " rare ", " summer is strong ", " 96-100 ", " ZLR3 ", " ZLR4 ", " ZLR5 ", " ZLR6 ",
" ZLR7 ", " ZLR8 ", " ZLR9 ", " ZLR10 ", " ZLR11 ", " ZLR12 ", " ZLR13 ", " gold early raw ", " 21-3 ", " ZLS1 ",
“ZLS2”、“ZLS3”、“ZLS4”、“01-20”、“87-534”、“ZLS5”、“ZLS6”、“ZLS7”、“ZLS8”、“ZLS9”、
" ZLS10 ", " ZLS11 ", " ZLS12 " seedling genomic DNA.DNA extraction method is referring to Murray MG, Thompson WF
(1980)Rapid isolation ofhigh molecular weight plant DNA.Nucleic Acids Res 8:
4321-4325。
2, amplification situation of the identification primers F rg13-F/Frg13-R in wild cabbage material
For amplification situation of the identification primer in wild cabbage, the synthesis of commission Shanghai JaRa biotechnology Services Co., Ltd
Primers F rg13-F and Frg13-R described in embodiment 1, or kit as described in example 2 is used, continue following steps:
The genomic DNA obtained using step 1 carries out PCR reaction as template, according to following reaction system and response procedures.
The PCR reaction system are as follows: 0.2 μ L/ μ L of templet gene group DNA includes Mg2+10 × PCR buffer buffering
0.1 μ L/ μ L, dNTPs 0.2mM, Taq archaeal dna polymerase 0.5U/ μ L of liquid, forward primer and 0.4 μM of reverse primer, remaining is double
Steam water.
Specific 10 μ L of reaction system total volume, wherein 2.0 μ L, 10 × PCR buffer buffer of templet gene group DNA
It (include Mg2+) 1 0.8 μ L, Taq DNA polymerase of μ L, dNTPs (2.5mM), 0.2 μ L (2.5U/ μ L), forward primer and anti-
To primer (10 μM) each 0.4 μ L, sterilize 5.2 μ L of ddH2O.
PCR response procedures are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 52-62 DEG C (52 DEG C, 55 DEG C, 58 DEG C, 60 DEG C,
62 DEG C) 30s, 72 DEG C of extension 45s, 35 circulations；72 DEG C of extension 7min；4 DEG C of preservations.
Amplified production electrophoresis detection in polyacrylamide gel, 160V constant pressure 70min, is clapped after fixation, silver staining, colour developing
According to.
The result shows that all wild cabbage samples are equal when primers F rg13-F/Frg13-R is used for wild cabbage selfing based material amplification
Non-specific amplification (Fig. 1) does not occur, band is clear, and difference is obvious.Assisting sifting is marked according to the method described above, reflects in seedling stage
31 plant are made with fusarium wilt disease resistance, this reaches 96% with the identical rate of post incoulation qualification result.
Embodiment 4, the verifying of the stability of primers F rg13-F/Frg13-R
Primers F rg13-F/Frg13-R described in embodiment 1 or kit as described in example 2 are adopted to wild cabbage self-mating system
" ZLR1 ", " ZLR2 ", " rare ", " summer is strong ", " 96-100 ", " ZLR3 ", " ZLR4 ", " ZLR5 ", " ZLR6 ", " ZLR7 ",
" ZLR8 ", " ZLR9 ", " ZLR10 ", " ZLR11 ", " ZLR12 ", " ZLR13 ", " gold early raw ", " 21-3 ", " ZLS1 ", " ZLS2 ",
“ZLS3”、“ZLS4”、“01-20”、“87-534”、“ZLS5”、“ZLS6”、“ZLS7”、“ZLS8”、“ZLS9”、“ZLS10”、
" ZLS11 ", " ZLS12 " are expanded, and in order to verify the stability of primer, we devise 4 within the temperature range of 53-62 DEG C
A temperature gradient carries out PCR according to following reaction system and program, compares steady when primer expands in wild cabbage selfing based material
It is qualitative.
PCR reaction system are as follows: 10 μ L of reaction system total volume, wherein 2.0 μ L, 10 × PCR of templet gene group DNA
Buffer buffer (includes Mg2+) 1 0.8 μ L, Taq DNA polymerase of μ L, dNTPs (2.5mM), 0.2 μ L (2.5U/ μ L),
Forward primer and reverse primer (10 μM) each 0.4 μ L, sterilize 5.2 μ L of ddH2O.
PCR response procedures are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 53-62 DEG C (53 DEG C, 56 DEG C, 59 DEG C, 62 DEG C)
30s, 72 DEG C of extension 45s, 35 circulations；72 DEG C of extension 7min；4 DEG C of preservations.
As a result as shown in table 1 and Fig. 2, it is warm no matter primers F rg13-F/Frg13-R anneals either one or two of between 53-62 DEG C
Non-specific amplification is not present in degree.Above-mentioned comparative test further demonstrates that primers F rg13-F/Frg13-R stability is good, special
Property is strong.
Shadow of the 1. different temperatures gradient of table to primers F rg13-F/Frg13-R specific amplification in wild cabbage selfing based material
It rings:
'+' there are non-specific amplification, '-', and non-specific amplification is not present.
Claims (9)
1. the PCR primer for screening wild cabbage (Brassica oleracea L.) fusarium wilt disease resistance material, nucleotide sequence
Are as follows:
Upstream primer Frg13-F:5`-ACCAGAGGCAGTTTTGGTTG-3 '
Downstream primer Frg13-R:5`-TCTTGCAACCCATGTCAAAA-3 '；
The Cabbage Wilt Disease resistant material characteristic bands size of the PCR primer amplification is 263bp, nucleotide sequence such as Seq
Shown in ID No.3.
2. the kit for screening Cabbage Wilt Disease resistant material, which is characterized in that draw including PCR described in claim 1
Object.
3. kit according to claim 2, which is characterized in that further include for examination needed for carrying out PCR and/or electrophoresis
Agent.
4. the method for screening Cabbage Wilt Disease resistant material, which is characterized in that including using PCR described in claim 1
Primer and/or kit described in claim 2 or 3 carry out following steps:
(1) PCR is carried out using genomic DNA of the PCR primer to wild cabbage material to be measured；
(2) PCR product described in detected through gel electrophoresis；
(3) it is filtered out from the electrophoresis detection result and the Cabbage Wilt Disease resistant material characteristic bands material of the same size
Material；
The Cabbage Wilt Disease resistant material characteristic bands are 263bp, and nucleotide sequence is as shown in Seq ID No.3.
5. according to the method described in claim 4, it is characterized in that, the PCR reaction system are as follows: 0.2 μ of templet gene group DNA
L/ μ L includes Mg2+10 × PCR buffer buffer, 0.1 μ L/ μ L, dNTPs 0.2mM, Taq archaeal dna polymerase 0.5U/ μ L,
Forward primer and 0.4 μM of reverse primer, remaining is distilled water.
6. according to the method described in claim 4, it is characterized in that, the reaction condition of the PCR are as follows: 94 DEG C of initial denaturation 5min；
94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 45s, 35 recycle；72 DEG C of extension 7min；4 DEG C of preservations.
7. according to the method described in claim 4, it is characterized in that, the detected through gel electrophoresis refers to the polyacrylamide using 8%
Amine gel is separated by electrophoresis 70 minutes in 160V invariable power, last silver staining colour developing.
8. the preparation method of kit described in claim 2 or 3, which is characterized in that indicating Cabbage Wilt Disease resistance screening
The packing box of purposes is provided with PCR primer described in claim 1.
9. the preparation method of kit according to any one of claims 8, which is characterized in that described to indicate Cabbage Wilt Disease resistance screening use
It is also equipped in the packing box on way for reagent needed for carrying out PCR and/or electrophoresis.
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| CN112266970B (en) * | 2020-07-22 | 2022-07-05 | 中国农业科学院蔬菜花卉研究所 | PCR primer and kit for cabbage black rot resistance screening and application of PCR primer and kit |
| CN111705157B (en) * | 2020-07-24 | 2022-11-29 | 中国农业科学院蔬菜花卉研究所 | PCR marker and primer for screening I-type S haplotypes of cabbage self-incompatible line |
| CN112980984A (en) * | 2021-02-19 | 2021-06-18 | 中国农业科学院蔬菜花卉研究所 | PCR primer for identifying physiological race types of pathogenic bacteria of cabbage wilt and application thereof |
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| CN102108407A (en) * | 2010-12-20 | 2011-06-29 | 北京市农林科学院 | Molecular marker and specific primers for assisting in test of wilt disease resistance in brassica oleracea and use thereof |
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| WO2001096549A1 (en) * | 2000-06-13 | 2001-12-20 | Scigen Harvest Co., Ltd. | Genes for s-adenosyl l-methionine: jasmonic acid carboxyl methyltransferase and a method for the development of pathogen- and stress-resistant plants using the genes |
| CN102108407A (en) * | 2010-12-20 | 2011-06-29 | 北京市农林科学院 | Molecular marker and specific primers for assisting in test of wilt disease resistance in brassica oleracea and use thereof |
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