CN109295244A - A kind of InDel primer and its application for distinguishing head cabbage varieties - Google Patents
A kind of InDel primer and its application for distinguishing head cabbage varieties Download PDFInfo
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Abstract
The invention discloses a kind of for distinguishing the InDel primer of head cabbage varieties, belongs to molecular biology molecule marker field.5 pairs of InDel primers that this method is filtered out by design, its nucleotide sequence is as shown in NO:1~5 SEQ ID, for expanding the DNA of unknown head cabbage varieties, passes through the bands of a spectrum after statistics gel electrophoresis, the kind with unique specificity bands of a spectrum is distinguished, and establishes the tree-like identification figure for being distinguished kind.Primer of the invention improves the stability of InDel reaction, it is easy to operate, efficiently and accurately, save primer, only easily 36 head cabbage varieties such as " QB1 " quickly can be distinguished on a molecular scale by 5 PCR, obtained cultivar identification figure has more intuitive than clustering tree, i.e., the primer that can distinguish any two kind can be rapidly found out according to cultivar identification figure, wild cabbage seedling early stage identification may be implemented in this method, also has extensive versatility on other species.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of to utilize the quick area of InDel molecular marking technique
Divide the method for head cabbage varieties.
Background technique
Wild cabbage is one of the main vegetables crop planted extensively of the world, and cultivation history is long, variety source and its abundant,
The every annual planting area of China cabbage accounts for the 25%~30% of national vegetable cultivation area, is at 400,000 hectares or more at present
The area spring such as northeast, northwest, North China, the summer, autumn culture main vegetables, also have big product cultivation in south.In recent years, with
The significantly extension of China's wild cabbage yield and cultivated area, more and more excellent variety are applied in Cabbage production.Therefore, it builds
The fast and reliable identification technology system of vertical head cabbage varieties is for the research protection of head cabbage varieties genetic resources, seedling stage assay, kind
Differentiation or even the sustainable development of wild cabbage industry etc. have great importance.
DNA molecular marker is one kind heredity generated as the biotechnologys such as molecular cloning and recombinant DNA rapidly develop
Label, since it is directly established on a molecular scale, without by the factors shadow such as external environment, Crop stage, sampling point
It rings, the polymorphic site of detection is unlimited, therefore qualification result has high reliability, and repeatability is high, and taste is strong.
Since traditional single morphology cultivar identification method has been unable to satisfy effectively identification at present or differentiation is current increasingly
The requirement of the kind increased.The application of molecular labeling brings great variety to Crop Genetic Breeding research, is also applied to simultaneously
In the research of cultivar identification.Not with numerous molecular marking techniques such as traditional DNA marker such as RAPD, ISSR, SRAP and AFLP
Together, InDel is labeled as the molecular marking technique of new generation developed based on complete genome DNA sequence, and InDel polymorphism is gene
A kind of two equipotential gene genetics label of specific type, shows as that different size of small pieces are inserted into or lacked in genome in group
Duan Xulie belongs to codominant marker, because it has many advantages, such as that preferable stability and polymorphism have been widely used in finger at present
Line map construction, the Study on Genetic Basis of germ plasm resource, the quick positioning of gene, high density gene linkage label, genetic diversity
Property analysis, assortment research etc..
It is existing to be distinguished in the research work with Germplasm Identification using molecular marking technique in progress vegetable crop kind, it is more
Digitized battlefield environment is drawn using computer software and obtains clustering tree in conjunction with statistics software clustering, but this is clustered
Analysis result not only can not intuitively show the primer for distinguishing kind, can not show discrimination process, and operating quantity is big, lack practical
Property.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of InDel primers for for distinguishing head cabbage varieties.
The present invention also technical problems to be solved are to provide above-mentioned primer and are distinguishing the application in head cabbage varieties.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
It is a kind of for distinguishing the InDel primer of head cabbage varieties, including following primer:
InDel22:F:aacacctgaaaccctcac；R:tttgaaagtcacccgtaa
InDel 24:F:actcctcccaacggtctt；R:gaacaggtggcacaagat
InDel 5:F:ttttggaaacggtcttgt；R:agcgactgaatggcaccc
InDel 3:F:aagcaaatgagcagaatga；R:tttgggctgtttaggtgg
InDel 34:F:tgggtcagttgtccaaag；R:tcagtacgcatatcagcac.
The above-mentioned InDel primer for distinguishing head cabbage varieties is distinguishing the application in head cabbage varieties.
The head cabbage varieties include: QB1, QB2, QB3, QB7, QB10, QB12, QB13, QB18, QB19, QB23,
QB28、QB30、QB37、QB39、QB40、QB41、QB43、QB46、QB90、QB100、 QB103、QB107、QB111、QB120、
QB121、QB168、QB171、QB185、QB 410、QB JF、 QBYSGL、QB142、QB161、QB179、QB158、QB197。
It is as follows that mirror method for distinguishing is carried out using 5 pairs of primer pair head cabbage varieties in the present invention:
(1) cabbage leaves genomic DNA is extracted；
(2) InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, obtained PCR is produced
Object is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 400bp, and in 280bp atypism bands of a spectrum；Benefit
Continuing InDel amplification with InDel24 primer, obtained PCR product is detected with polyacrylamide gel electrophoresis, if
Occur characteristic spectrum belt at 280bp, and the atypism bands of a spectrum at 450bp；It continues with InDel5 primer and carries out InDel expansion
Increase, obtained PCR product is detected with polyacrylamide gel electrophoresis, if occurring characteristic at 100bp, 150bp, 350bp
Bands of a spectrum recycle InDel3 primer to continue InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis,
If occurring characteristic spectrum belt at 450bp, kind QB2, if occurring characteristic spectrum belt at 400bp, kind is
QB28, if occurring characteristic spectrum belt at 500bp, kind QB107；
When carrying out InDel amplification using InDel24 primer, if occurring characteristic spectrum belt at 280bp, 450bp；Continue
Continuing InDel using InDel5 primer to expand, obtained PCR product is detected with polyacrylamide gel electrophoresis, if
Occur characteristic spectrum belt at 350bp, and does not occur characteristic spectrum belt in 100bp, 150bp, kind QB1, if
Occur characteristic spectrum belt at 100bp, 150bp, 350bp, recycles InDel3 primer to continue InDel amplification, obtain
PCR product is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 300bp, 450bp, kind QB18,
If occurring characteristic spectrum belt at 300bp, 600bp, kind QB111；
(3) InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, if at 280bp
There is characteristic spectrum belt, and in 400bp atypism bands of a spectrum；Continue InDel using InDel24 primer to expand, obtain
PCR product is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 280bp, and without feature at 450bp
Property bands of a spectrum；It continues with InDel5 primer and continues InDel amplification, obtained PCR product polyacrylamide gel electrophoresis
Detection, if occurring characteristic spectrum belt at 100bp, 350bp, kind QB197；If the atypism bands of a spectrum at 350bp,
And there is characteristic spectrum belt, kind QBQ168 in 400bp；If the atypism bands of a spectrum at 100bp, which is
QB120；If occurring characteristic spectrum belt at 400bp, 350bp, which is QB90；If occurring characteristic spectrum at 450bp
Band, the kind are QB171；If occurring characteristic spectrum belt at 100bp, 150bp, 300bp, InDel3 primer is continued with
Continue InDel amplification, obtained PCR product is detected with polyacrylamide gel electrophoresis；If going out at 300bp, 500bp
Existing characteristic spectrum belt, kind QB43；If occurring characteristic spectrum belt at 480bp, kind QB41；If 300bp,
Occur characteristic spectrum belt, kind QB13 at 400bp；If occurring characteristic spectrum belt at 400bp, 450bp, utilize
InDel34 primer continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if in 100bp
There is characteristic spectrum belt, kind QB12 in place；If the atypism bands of a spectrum at 100bp, kind QB103；
InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, if occurring at 280bp
Characteristic spectrum belt, and in 400bp atypism bands of a spectrum；Continue InDel using InDel24 primer to expand, obtained PCR is produced
Object is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 280bp, 450bp；InDel5 is continued with to draw
Object continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if in 350bp, 450bp, place
There is characteristic spectrum belt, kind QB179；If occurring characteristic spectrum belt at 350bp, kind QB161；If
Atypism bands of a spectrum at 100bp, and have characteristic spectrum belt at 150bp, 350bp, which is QB46；If going out at 400bp
Existing characteristic spectrum belt, the kind are QB30；If occurring characteristic spectrum belt at 100bp, 150bp, 350bp, continue with
InDel3 primer continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis；If 600bp,
Occur characteristic spectrum belt, kind QB3 at 300bp；If occurring characteristic spectrum belt at 480bp, 500bp, kind is
QB39；If occurring characteristic spectrum belt at 300bp, 450bp, continues InDel using InDel34 primer and expand, obtain
PCR product detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 400bp, kind QB19；If
The atypism bands of a spectrum at 400bp, kind QB121；If the equal atypism bands of a spectrum at 300bp, 500bp, utilize
InDel34 primer continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if at 420bp
Appearance its kind of characteristic spectrum belt is QB410；If not occurring its kind of characteristic spectrum belt at 420bp is QB142；
(4) genomic DNA obtained using InDel22 primer pair step (1) carries out InDel amplification, if 280bp,
Occurs characteristic spectrum belt at 400bp；Continue InDel using InDel24 primer to expand, obtained PCR product is with poly- third
Acrylamide detected through gel electrophoresis, if occurring characteristic spectrum belt at 280bp, and the atypism bands of a spectrum at 450bp；It continues with
InDel5 primer continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if at 400bp
There is signature band, kind QB37；If occurring characteristic spectrum belt at 100bp, 150bp, 350bp, utilize
InDel3 primer continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if at 300bp
There is characteristic spectrum belt, kind QB10；If occurring characteristic spectrum belt at 250bp, kind QB40；If 400bp,
Occur characteristic spectrum belt, kind QBJF at 450bp；
InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, if in 280bp, 400bp
There is characteristic spectrum belt in place；Continue InDel using InDel24 primer to expand, obtained PCR product is solidifying with polyacrylamide
Gel electrophoresis detection, if occurring characteristic spectrum belt at 280bp, 450bp；It continues with InDel5 primer and continues InDel
Amplification, obtained PCR product is detected with polyacrylamide gel electrophoresis, if the atypism bands of a spectrum at 100bp, and 150bp,
Occur characteristic spectrum belt, kind QB100 at 300bp；If the atypism bands of a spectrum at 350bp, and occur at 400bp special
Sign property bands of a spectrum, kind QB185；If occurring characteristic spectrum belt at 100bp, 150bp, 350bp, drawn using InDel3
Object continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis；If occurring feature at 300bp
Property bands of a spectrum, kind QB7；If occurring characteristic spectrum belt at 300bp, 450bp, kind QB23；If 350bp,
Occur characteristic spectrum belt at 400bp, continues InDel using InDel3 primer and expand, obtained PCR product is with poly- third
Acrylamide detected through gel electrophoresis, if occurring characteristic spectrum belt at 250bp, 300bp, 400bp, using InDel34 primer after
Continuous to carry out InDel amplification, obtained PCR product is detected with polyacrylamide gel electrophoresis；If occurring characteristic spectrum belt at 250
Its kind is QBYSGL；If not occurring its kind of characteristic spectrum belt at 250 is QB158.
In step (1), the method for extracting cabbage leaves genomic DNA is as follows:
Wild cabbage young leaflet tablet 0.1g is taken, through liquid nitrogen grinding, 500 μ l CTAB lysates is added, are transferred in 1.5ml centrifuge tube,
Cooling is taken out after 65 DEG C of 0.5~1h of water-bath, is jiggled after isometric chloroform/iso pentane alcohol mixture is added, 12000r/min,
It is centrifuged 8~10min, supernatant is extracted 1~2 time with chloroform/iso pentane alcohol mixture, and pre- cold isopropanol is added, quiet under the conditions of 4 DEG C
It sets 3 hours or more, collects the ethanol washing that cotton-shaped DNA volume fraction is 70%, cotton-shaped DNA is dissolved in TE buffer after drying
Middle preservation；
Wherein, the composition of the CTAB lysate is as follows: volume fraction 2%CTAB, 2mol/L NaCl2,
20mmol/L EDTA, 100mmol/L Tris-HCl, PH=8.0, volume fraction is 0.2% beta -mercaptoethanol；The chlorine
The volume ratio of chloroform and isoamyl alcohol is 24:1 in imitative/iso pentane alcohol mixture.
In step (2)~(5), the InDel amplification, PCR reaction system is 20 μ l, wherein containing genomic DNA 1
μ l, each 10 μ l of 1 μ l, 2xEasyTaq PCR SuperMix for PAGE of upstream and downstream primer.
In step (2)~(5), the InDel amplification, PCR reaction condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of changes
Property 50S, 56 DEG C of 50S, 72 DEG C of extension 60S, 35 circulation；Then 72 DEG C of extension 10min are saved in 4 DEG C；
Wherein, the annealing temperature of InDel22 primer is 46.7 DEG C, and the annealing temperature of InDel24 is 47.1 DEG C, InDel5's
Annealing temperature is 48.4 DEG C, and the annealing temperature of InDel3 is 46.5 DEG C；The annealing temperature of InDel34 is 49.1 DEG C.
The utility model has the advantages that
Primer of the invention improves the stability of InDel reaction, and easy to operate, efficiently and accurately is saved primer, only passed through
5 times PCR easily can quickly be distinguished 36 head cabbage varieties such as " QB1 " on a molecular scale, obtained kind mirror
Figure is determined than clustering tree with more intuitive, i.e., can rapidly find out according to cultivar identification figure can distinguish drawing for any two kind
Object, this method may be implemented wild cabbage seedling early stage identification, also have extensive versatility on other species.
Detailed description of the invention
Fig. a kind of the present invention and number comparative diagram.
Fig. 2 present invention identifies arborescence.
Fig. 3 present invention identifies arborescence.
Fig. 4 present invention identifies arborescence.
Fig. 5 present invention identifies arborescence.
Fig. 6 present invention identifies arborescence.
Fig. 7 present invention identifies arborescence
Fig. 8 utilizes InDel22 primer PCR product polypropylene acrylamide gel electrophoretogram.
Fig. 9 utilizes InDel24 primer PCR product polypropylene acrylamide gel electrophoretogram.
Figure 10 utilizes InDel5 primer PCR product polypropylene acrylamide gel electrophoretogram.
Figure 11 utilizes InDel3 primer PCR product polypropylene acrylamide gel electrophoretogram.
Figure 12 utilizes InDel34 primer PCR product polypropylene acrylamide gel electrophoretogram.
Specific embodiment
Embodiment 1: the method for cabbage leaves genomic DNA is as follows:
Wild cabbage young leaflet tablet 0.1g is taken, through liquid nitrogen grinding, 500 μ l CTAB lysates is added, are transferred in 1.5ml centrifuge tube,
Cooling is taken out after 65 DEG C of 0.5~1h of water-bath, is jiggled after isometric chloroform/iso pentane alcohol mixture is added, 12000r/
Min is centrifuged 8~10min, and supernatant is extracted 1~2 time with chloroform/iso pentane alcohol mixture, pre- cold isopropanol is added, in 4 DEG C of conditions
Lower standing 3 hours or more, the ethanol washing that cotton-shaped DNA volume fraction is 70% is collected, cotton-shaped DNA is dissolved in TE after dry and is delayed
It is saved in fliud flushing；
Wherein, the composition of the CTAB lysate is as follows: volume fraction 2%CTAB, 2mol/L NaCl2,
20mmol/L EDTA, 100mmol/L Tris-HCl, PH=8.0, volume fraction is 0.2% beta -mercaptoethanol；The chlorine
The volume ratio of chloroform and isoamyl alcohol is 24:1 in imitative/iso pentane alcohol mixture.
The amplification of InDel described in step (2)~(5), PCR reaction system is 20 μ l, wherein the 1 μ l containing genomic DNA,
Each 10 μ l of 1 μ l, 2xEasyTaq PCR SuperMix for PAGE of upstream and downstream primer.
The amplification of InDel described in step (2)~(5), PCR reaction condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation
50S, 56 DEG C of 50S, 72 DEG C of extension 60S, 35 circulations；Then 72 DEG C of extension 10min are saved in 4 DEG C；
Wherein, the annealing temperature of InDel22 primer is 46.7 DEG C, and the annealing temperature of InDel24 is 47.1 DEG C, InDel5's
Annealing temperature is 48.4 DEG C, and the annealing temperature of InDel13 is 46.5 DEG C.
Embodiment 2:
The head cabbage varieties that the present invention can distinguish include:
QB1、QB2、QB3、QB7、QB10、QB12、QB13、QB18、QB19、QB23、QB28、 QB30、QB37、QB39、
QB40、QB41、QB43、QB46、QB90、QB100、QB103、QB107、QB111、QB120、QB121、QB168、QB171、
QB185、QB 410、QB JF、QBYSGL、QB142、 QB161、QB179、QB158、QB197。
(1) cabbage leaves genomic DNA is extracted；
(2) InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, obtained PCR is produced
Object is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 400bp, and in 280bp atypism bands of a spectrum；Benefit
Continuing InDel amplification with InDel24 primer, obtained PCR product is detected with polyacrylamide gel electrophoresis, if
Occur characteristic spectrum belt at 280bp, and the atypism bands of a spectrum at 450bp；It continues with InDel5 primer and carries out InDel expansion
Increase, obtained PCR product is detected with polyacrylamide gel electrophoresis, if occurring characteristic at 100bp, 150bp, 350bp
Bands of a spectrum recycle InDel3 primer to continue InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis,
If occurring characteristic spectrum belt at 450bp, kind QB2, if occurring characteristic spectrum belt at 400bp, kind is
QB28, if occurring characteristic spectrum belt at 500bp, kind QB107；
When carrying out InDel amplification using InDel24 primer, if occurring characteristic spectrum belt at 280bp, 450bp；Continue
Continuing InDel using InDel5 primer to expand, obtained PCR product is detected with polyacrylamide gel electrophoresis, if
Occur characteristic spectrum belt at 350bp, and does not occur characteristic spectrum belt in 100bp, 150bp, kind QB1, if
Occur characteristic spectrum belt at 100bp, 150bp, 350bp, recycles InDel3 primer to continue InDel amplification, obtain
PCR product is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 300bp, 450bp, kind QB18,
If occurring characteristic spectrum belt at 300bp, 600bp, kind QB111；
(3) InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, if at 280bp
There is characteristic spectrum belt, and in 400bp atypism bands of a spectrum；Continue InDel using InDel24 primer to expand, obtain
PCR product is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 280bp, and without feature at 450bp
Property bands of a spectrum；It continues with InDel5 primer and continues InDel amplification, obtained PCR product polyacrylamide gel electrophoresis
Detection, if occurring characteristic spectrum belt at 100bp, 350bp, kind QB197；If the atypism bands of a spectrum at 350bp,
And there is characteristic spectrum belt, kind QBQ168 in 400bp；If the atypism bands of a spectrum at 100bp, which is
QB120；If occurring characteristic spectrum belt at 400bp, 350bp, which is QB90；If occurring characteristic spectrum at 450bp
Band, the kind are QB171；If occurring characteristic spectrum belt at 100bp, 150bp, 300bp, InDel3 primer is continued with
Continue InDel amplification, obtained PCR product is detected with polyacrylamide gel electrophoresis；If going out at 300bp, 500bp
Existing characteristic spectrum belt, kind QB43；If occurring characteristic spectrum belt at 480bp, kind QB41；If 300bp,
Occur characteristic spectrum belt, kind QB13 at 400bp；If occurring characteristic spectrum belt at 400bp, 450bp, utilize
InDel34 primer continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if in 100bp
There is characteristic spectrum belt, kind QB12 in place；If the atypism bands of a spectrum at 100bp, kind QB103；
InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, if occurring at 280bp
Characteristic spectrum belt, and in 400bp atypism bands of a spectrum；Continue InDel using InDel24 primer to expand, obtained PCR is produced
Object is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 280bp, 450bp；InDel5 is continued with to draw
Object continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if in 350bp, 450bp, place
There is characteristic spectrum belt, kind QB179；If occurring characteristic spectrum belt at 350bp, kind QB161；If
Atypism bands of a spectrum at 100bp, and have characteristic spectrum belt at 150bp, 350bp, which is QB46；If going out at 400bp
Existing characteristic spectrum belt, the kind are QB30；If occurring characteristic spectrum belt at 100bp, 150bp, 350bp, continue with
InDel3 primer continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis；If 600bp,
Occur characteristic spectrum belt, kind QB3 at 300bp；If occurring characteristic spectrum belt at 480bp, 500bp, kind is
QB39；If occurring characteristic spectrum belt at 300bp, 450bp, continues InDel using InDel34 primer and expand, obtain
PCR product detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 400bp, kind QB19；If
The atypism bands of a spectrum at 400bp, kind QB121；If the equal atypism bands of a spectrum at 300bp, 500bp, utilize
InDel34 primer continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if at 420bp
Appearance its kind of characteristic spectrum belt is QB410；If not occurring its kind of characteristic spectrum belt at 420bp is QB142；
(4) genomic DNA obtained using InDel22 primer pair step (1) carries out InDel amplification, if 280bp,
Occurs characteristic spectrum belt at 400bp；Continue InDel using InDel24 primer to expand, obtained PCR product is with poly- third
Acrylamide detected through gel electrophoresis, if occurring characteristic spectrum belt at 280bp, and the atypism bands of a spectrum at 450bp；It continues with
InDel5 primer continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if at 400bp
There is signature band, kind QB37；If occurring characteristic spectrum belt at 100bp, 150bp, 350bp, utilize
InDel3 primer continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if at 300bp
There is characteristic spectrum belt, kind QB10；If occurring characteristic spectrum belt at 250bp, kind QB40；If 400bp,
Occur characteristic spectrum belt, kind QBJF at 450bp；
InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, if in 280bp, 400bp
There is characteristic spectrum belt in place；Continue InDel using InDel24 primer to expand, obtained PCR product is solidifying with polyacrylamide
Gel electrophoresis detection, if occurring characteristic spectrum belt at 280bp, 450bp；It continues with InDel5 primer and continues InDel
Amplification, obtained PCR product is detected with polyacrylamide gel electrophoresis, if the atypism bands of a spectrum at 100bp, and 150bp,
Occur characteristic spectrum belt, kind QB100 at 300bp；If the atypism bands of a spectrum at 350bp, and occur at 400bp special
Sign property bands of a spectrum, kind QB185；If occurring characteristic spectrum belt at 100bp, 150bp, 350bp, drawn using InDel3
Object continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis；If occurring feature at 300bp
Property bands of a spectrum, kind QB7；If occurring characteristic spectrum belt at 300bp, 450bp, kind QB23；If 350bp,
Occur characteristic spectrum belt at 400bp, continues InDel using InDel3 primer and expand, obtained PCR product is with poly- third
Acrylamide detected through gel electrophoresis, if occurring characteristic spectrum belt at 250bp, 300bp, 400bp, using InDel34 primer after
Continuous to carry out InDel amplification, obtained PCR product is detected with polyacrylamide gel electrophoresis；If occurring characteristic spectrum belt at 250
Its kind is QBYSGL；If not occurring its kind of characteristic spectrum belt at 250 is QB158.
Sequence table
<110>Jiangsu Province Agriculture Science Institute
<120>a kind of InDel primer and its application for distinguishing head cabbage varieties
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aacacctgaa accctcac 18
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tttgaaagtc acccgtaa 18
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
actcctccca acggtctt 18
<210> 4
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gaacaggtgg cacaagat 18
<210> 5
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ttttggaaac ggtcttgt 18
<210> 6
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
agcgactgaa tggcaccc 18
<210> 7
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
aagcaaatga gcagaatga 19
<210> 8
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tttgggctgt ttaggtgg 18
<210> 9
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
tgggtcagtt gtccaaag 18
<210> 10
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
tcagtacgca tatcagcac 19
Claims (7)
1. a kind of for distinguishing the InDel primer of head cabbage varieties, which is characterized in that including following primer:
InDel22:F:aacacctgaaaccctcac；R:tttgaaagtcacccgtaa
InDel 24:F:actcctcccaacggtctt；R:gaacaggtggcacaagat
InDel 5:F:ttttggaaacggtcttgt；R:agcgactgaatggcaccc
InDel 3:F:aagcaaatgagcagaatga；R:tttgggctgtttaggtgg
InDel 34:F:tgggtcagttgtccaaag；R:tcagtacgcatatcagcac.
2. the InDel primer described in claim 1 for distinguishing head cabbage varieties is distinguishing the application in head cabbage varieties.
3. application according to claim 2, which is characterized in that the head cabbage varieties include: QB1, QB2, QB3, QB7,
QB10、QB12、QB13、QB18、QB19、QB23、QB28、QB30、QB37、QB39、QB40、QB41、QB43、QB46、QB90、
QB100、QB103、QB107、QB111、QB120、QB121、QB168、QB171、QB185、QB 410、QB JF、QBYSGL、
QB142、QB161、QB179、QB158、QB197。
4. application according to claim 2, spy's card is, include the following steps:
(1) cabbage leaves genomic DNA is extracted；
(2) InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, obtained PCR product is used poly-
Acrylamide gel electrophoresis detection, if occurring characteristic spectrum belt at 400bp, and in 280bp atypism bands of a spectrum；It utilizes
InDel24 primer continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if in 280bp
There is characteristic spectrum belt in place, and the atypism bands of a spectrum at 450bp；It continues with InDel5 primer and carries out InDel amplification, obtain
PCR product detected with polyacrylamide gel electrophoresis, if occur characteristic spectrum belt at 100bp, 150bp, 350bp, then
Continuing InDel using InDel3 primer to expand, obtained PCR product is detected with polyacrylamide gel electrophoresis, if
Occur characteristic spectrum belt at 450bp, kind QB2, if occurring characteristic spectrum belt at 400bp, kind QB28, if
Occur characteristic spectrum belt, kind QB107 at 500bp；
When carrying out InDel amplification using InDel24 primer, if occurring characteristic spectrum belt at 280bp, 450bp；It continues with
InDel5 primer continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if at 350bp
There is characteristic spectrum belt, and do not occur characteristic spectrum belt in 100bp, 150bp, kind QB1, if 100bp,
Occur characteristic spectrum belt at 150bp, 350bp, InDel3 primer is recycled to continue InDel amplification, obtained PCR product
It is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 300bp, 450bp, kind QB18, if
Occur characteristic spectrum belt, kind QB111 at 300bp, 600bp；
(3) InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, if occurring at 280bp special
Sign property bands of a spectrum, and in 400bp atypism bands of a spectrum；Continue InDel using InDel24 primer to expand, obtained PCR product
It is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 280bp, and the atypism bands of a spectrum at 450bp；After
Continuous to continue InDel amplification using InDel5 primer, obtained PCR product is detected with polyacrylamide gel electrophoresis, if
Occur characteristic spectrum belt, kind QB197 at 100bp, 350bp；If the atypism bands of a spectrum at 350bp, and in 400bp
There is characteristic spectrum belt, kind QBQ168；If the atypism bands of a spectrum at 100bp, which is QB120；If
Occurs characteristic spectrum belt at 400bp, 350bp, which is QB90；If occurring characteristic spectrum belt at 450bp, which is
QB171；If occurring characteristic spectrum belt at 100bp, 150bp, 300bp, continues with InDel3 primer and continue InDel
Amplification, obtained PCR product are detected with polyacrylamide gel electrophoresis；If occurring characteristic spectrum belt at 300bp, 500bp,
Its kind is QB43；If occurring characteristic spectrum belt at 480bp, kind QB41；If occurring at 300bp, 400bp special
Sign property bands of a spectrum, kind QB13；If occurring characteristic spectrum belt at 400bp, 450bp, using InDel34 primer continue into
Row InDel amplification, obtained PCR product is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 100bp,
Its kind is QB12；If the atypism bands of a spectrum at 100bp, kind QB103；
InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, if occurring feature at 280bp
Property bands of a spectrum, and in 400bp atypism bands of a spectrum；Continue InDel using InDel24 primer to expand, obtained PCR product is used
Polyacrylamide gel electrophoresis detection, if occurring characteristic spectrum belt at 280bp, 450bp；Continue with InDel5 primer after
Continuous to carry out InDel amplification, obtained PCR product is detected with polyacrylamide gel electrophoresis, if going out at 350bp, 450bp, place
Existing characteristic spectrum belt, kind QB179；If occurring characteristic spectrum belt at 350bp, kind QB161；If in 100bp
Locate atypism bands of a spectrum, and have characteristic spectrum belt at 150bp, 350bp, which is QB46；If occurring feature at 400bp
Property bands of a spectrum, the kind be QB30；If occurring characteristic spectrum belt at 100bp, 150bp, 350bp, continues with InDel3 and draw
Object continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis；If at 600bp, 300bp
There is characteristic spectrum belt, kind QB3；If occurring characteristic spectrum belt at 480bp, 500bp, kind QB39；If
Occur characteristic spectrum belt at 300bp, 450bp, continues InDel using InDel34 primer and expand, obtained PCR product
It is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum belt at 400bp, kind QB19；If the nothing at 400bp
Characteristic spectrum belt, kind QB121；If the equal atypism bands of a spectrum at 300bp, 500bp, continued using InDel34 primer
Carry out InDel amplification, obtained PCR product detects with polyacrylamide gel electrophoresis, if occur at 420bp characteristic spectrum belt its
Kind is QB410；If not occurring its kind of characteristic spectrum belt at 420bp is QB142；
(4) InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, if at 280bp, 400bp
There is characteristic spectrum belt；Continue InDel using InDel24 primer to expand, obtained PCR product is solidifying with polyacrylamide
Gel electrophoresis detection, if occurring characteristic spectrum belt at 280bp, and the atypism bands of a spectrum at 450bp；InDel5 is continued with to draw
Object continues InDel amplification, and obtained PCR product is detected with polyacrylamide gel electrophoresis, if occurring feature at 400bp
Property band, kind QB37；If occurring characteristic spectrum belt at 100bp, 150bp, 350bp, using InDel3 primer after
Continuous to carry out InDel amplification, obtained PCR product is detected with polyacrylamide gel electrophoresis, if occurring characteristic spectrum at 300bp
Band, kind QB10；If occurring characteristic spectrum belt at 250bp, kind QB40；If going out at 400bp, 450bp
Existing characteristic spectrum belt, kind QBJF；
InDel amplification is carried out using the genomic DNA that InDel22 primer pair step (1) obtains, if going out at 280bp, 400bp
Existing characteristic spectrum belt；Continue InDel using InDel24 primer to expand, obtained PCR product polyacrylamide gel electricity
Swimming detection, if occurring characteristic spectrum belt at 280bp, 450bp；It continues with InDel5 primer and continues InDel amplification,
Obtained PCR product is detected with polyacrylamide gel electrophoresis, if the atypism bands of a spectrum at 100bp, and in 150bp, 300bp
There is characteristic spectrum belt, kind QB100 in place；If the atypism bands of a spectrum at 350bp, and occurs characteristic at 400bp
Bands of a spectrum, kind QB185；If occurring characteristic spectrum belt at 100bp, 150bp, 350bp, continued using InDel3 primer
InDel amplification is carried out, obtained PCR product is detected with polyacrylamide gel electrophoresis；If occurring characteristic spectrum at 300bp
Band, kind QB7；If occurring characteristic spectrum belt at 300bp, 450bp, kind QB23；If in 350bp, 400bp
There is characteristic spectrum belt in place, continues InDel using InDel3 primer and expands, obtained PCR product polyacrylamide
Detected through gel electrophoresis is continued if occurring characteristic spectrum belt at 250bp, 300bp, 400bp using InDel34 primer
InDel amplification, obtained PCR product are detected with polyacrylamide gel electrophoresis；If occurring its kind of characteristic spectrum belt at 250
For QBYSGL；If not occurring its kind of characteristic spectrum belt at 250 is QB158.
5. application according to claim 4, which is characterized in that step (1) extracts the method for cabbage leaves genomic DNA such as
Under:
Wild cabbage young leaflet tablet 0.1g is taken, through liquid nitrogen grinding, 500 μ l CTAB lysates is added, are transferred in 1.5ml centrifuge tube, 65 DEG C
Cooling is taken out after 0.5~1h of water-bath, is jiggled after isometric chloroform/iso pentane alcohol mixture is added, 12000r/min, is centrifuged
8~10min, supernatant are extracted 1~2 time with chloroform/iso pentane alcohol mixture, pre- cold isopropanol are added, stands 3 under the conditions of 4 DEG C
Hour or more, the ethanol washing that cotton-shaped DNA volume fraction is 70% is collected, cotton-shaped DNA is dissolved in TE buffer after dry
It saves；
Wherein, the composition of the CTAB lysate is as follows: volume fraction 2%CTAB, 2mol/L NaCl2, 20mmol/
LEDTA, 100mmol/L Tris-HCl, PH=8.0, volume fraction is 0.2% beta -mercaptoethanol；Chloroform/the isoamyl alcohol
The volume ratio of chloroform and isoamyl alcohol is 24:1 in mixture.
6. application according to claim 4, which is characterized in that the amplification of InDel described in step (2)~(5), PCR are anti-
Answering system is 20 μ l, wherein the 1 μ l containing genomic DNA, each 1 μ l, 2xEasyTaq PCR SuperMix for of upstream and downstream primer
PAGE 10μl。
7. application according to claim 4, which is characterized in that the amplification of InDel described in step (2)~(5), PCR are anti-
Answer condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 50S, 56 DEG C of 50S, 72 DEG C of extension 60S, 35 recycle；72 DEG C of extensions
Then 10min is saved in 4 DEG C；
Wherein, the annealing temperature of InDel22 primer is 46.7 DEG C, and the annealing temperature of InDel24 is 47.1 DEG C, the annealing of InDel5
Temperature is 48.4 DEG C, and the annealing temperature of InDel3 is 46.5 DEG C；The annealing temperature of InDel34 is 49.1 DEG C.
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| CN201810947779.6A CN109295244A (en) | 2018-08-20 | 2018-08-20 | A kind of InDel primer and its application for distinguishing head cabbage varieties |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528698A (en) * | 2021-07-14 | 2021-10-22 | 中国农业科学院油料作物研究所 | InDel molecular marker for identifying and/or distinguishing cabbage vegetables and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103224930A (en) * | 2013-04-12 | 2013-07-31 | 上海交通大学 | Brassica campestris L.ssp.chinensis SSR marker primer set and application of the same in variety identification |
-
2018
- 2018-08-20 CN CN201810947779.6A patent/CN109295244A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103224930A (en) * | 2013-04-12 | 2013-07-31 | 上海交通大学 | Brassica campestris L.ssp.chinensis SSR marker primer set and application of the same in variety identification |
Non-Patent Citations (1)
| Title |
|---|
| 杨双娟等: "利用Indel标记鉴定‘豫甘3号’结球甘蓝种子纯度", 《分子植物育种》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528698A (en) * | 2021-07-14 | 2021-10-22 | 中国农业科学院油料作物研究所 | InDel molecular marker for identifying and/or distinguishing cabbage vegetables and application thereof |
| CN113528698B (en) * | 2021-07-14 | 2022-01-25 | 中国农业科学院油料作物研究所 | InDel molecular marker for identifying and/or distinguishing cabbage vegetables and application thereof |
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