CN101178388A - Establishment of cabbage hybrid Xiaguang and Zaoxia 16 fingerprint - Google Patents
Establishment of cabbage hybrid Xiaguang and Zaoxia 16 fingerprint Download PDFInfo
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- CN101178388A CN101178388A CNA2006101182549A CN200610118254A CN101178388A CN 101178388 A CN101178388 A CN 101178388A CN A2006101182549 A CNA2006101182549 A CN A2006101182549A CN 200610118254 A CN200610118254 A CN 200610118254A CN 101178388 A CN101178388 A CN 101178388A
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Abstract
The invention discloses a DNA fingerprint chromatogram of cabbage hybrids and the parents, as well as an establishing method and application. The establishing method includes: 1) cabbage DNA is extracted and purified; 2) the RAPD analysis is carried out for a template by the obtained high-purity cabbage DNA; 3) the representative polymorphic RAPD amplification strips is selected to construct the DNA fingerprint chromatogram for testing materials; the 'Xiaguang' and 'Zaoxia 16' cabbage hybrids and the parents all have the specific DNA fingerprints in the DNA fingerprint chromatogram, which can be distinguished mutually. As the DNA fingerprint chromatogram of the invention is showed in the figure form, the chromatogram is more intuitive and easier to be understood; the DNA fingerprint chromatogram is converted to the digital expressing form, which facilitates the computer to read and analyze. The invention can carry out the purity identification of the seeds of 'Xiaguang' and 'Zaoxia 16' cabbage hybrids from the genetic natures, the results are accurate and reliable, and the detection is rapid.
Description
Technical field
The present invention relates to the cabbage seed authenticate technology, specifically the dna fingerprinting of wild cabbage and method for building up and application.
Background technology
At present, the master who applies in China's production plants wild cabbage (Brassica oleracea var.capitata) kind and is essentially the first generation of hybrid.The first generation of hybrid is many merits of parent comprehensively, better economic benefit.
But understand the defective of improper owing to control measures for self-incompatibility itself and usually produce selfed seed in production of hybrid seeds process, promptly pseudostationary causes seed purity to descend.Therefore, improving purity of hybrid is the important topic of heterosis utilization research field.At present, cultivar identification and seed purity analytical approach roughly have three kinds, i.e. land for growing field crops form, biochemical marker and dna fingerprinting identification method.Traditional land for growing field crops identification method with economical characters such as the color of histoorgans such as seed, seedling, leaf, flower, fruit, pollen, forms as the observation index of identifying, though be a kind of comparatively feasible method in production practices, but identifying, the land for growing field crops needs extra land occupation, cycle is long, cost is big, not only be subject to seasonal restrictions, and the performance of a lot of proterties is subjected to the influence of cultivation step and envirment factor, and assessor's observation experience is also restricting the accuracy of identifying.The land for growing field crops identifies that maximum defective is that seed purity information can not in time be provided, and can not conclude the business prerequisite in a large number for the quality information of relevant seed at seed, can not play the effect of supervision in advance to seed quality.Biochemical identification method comprises protein electrophorese and isozyme electrophoresis, has obtained increasingly extensive application, but can not show interracial polymorphism fully, is difficult to the nearer kind of sibship is effectively distinguished.
Summary of the invention
The purpose of this invention is to provide the method for building up and the application of a kind of cabbage hybrid " summer light ", " summer 16 early " finger-print, to overcome the deficiency that prior art exists.
Technical conceive of the present invention is such:
The dna fingerprinting identification method is the molecule labelling method based on the polymorphism of DNA, can remedy and overcome many defectives and a difficult problem in the morphology evaluation of seed purity and isodynamic enzyme, seed protein electrophoresis are identified.The present invention has utilized SRAP, RAPD technique construction middle and lower reach of Yangtze River area wild cabbage main breed " summer light ", " summer 16 early " and its dna fingerprinting of parent separately; can provide scientific basis for the evaluation and the purity detecting of these kinds, and then the protection producer and user's legitimate rights and interests.
Cabbage hybrid of the present invention " summer light ", " summer 16 early " and parent's thereof dna fingerprinting, by the data that " 1 " and " 0 " are formed, " summer light " maternal data are 10111100110111; The data of " summer light " are 11111111110111; The data of " summer light " male parent are 11111111011111; " summer 16 early ", maternal data were 10111111111101111; The data in " summer 16 early " are 11111011010010001; " summer 16 early " data of male parent are 11110101111100000.Have amplified band to exist on certain position in wherein said numeral " 1 " the expression collection of illustrative plates, digital " 0 " is illustrated in does not have amplified band to exist on certain position.
Said " summer light ", " summer 16 early " cabbage hybrid and parent can adopt the product of Inst. of Gardening, Academy of Agriculture of Shanghai's public offering;
The method for building up of wild cabbage dna fingerprinting comprises the steps:
1) extraction and the genomic DNA of purifying wild cabbage;
2) be that template is carried out the RAPD analysis with the wild cabbage DNA that obtains;
3) select representational polymorphism RAPD amplified band, having, not having and make up according to selected band for trying the material dna fingerprinting.
Wherein said " summer light ", " early summer 16 " cabbage hybrid and separately the parent its special dna fingerprint is all arranged, can make a distinction mutually.The primer that adopts in the described RAPD amplification is S42, is to give birth to the synthetic random oligonucleotide primer of worker's bioengineering company limited by Shanghai, and its base sequence is GGACCCAACC.
Dna fingerprinting of the present invention can be applied to the evaluation of " summer light " and " summer 16 early " cabbage hybrid seed purity.
The present invention has following advantage:
1. the present invention has created " summer light ", " summer 16 early " cabbage hybrid and parent's thereof dna fingerprinting, the result of study of cabbage seed being identified with the dna fingerprint analytical technology is disclosed, its method for building up can be identified cabbage seed from inheritance, therefore accurately, reliably, has legal effect.
2. employing the present invention, to " summer light " and " early summer 16 " when the cabbage seed sample truly detects, extract the DNA of testing sample with quick, the minim DNA extraction method of present widespread use, with the DNA that extracts as template, carrying out RAPD analyzes, in several hrs, just can know this dna fingerprint, therefore, just can judge its authenticity rapidly.
3. because dna fingerprinting of the present invention is the form tabular form with figure, seem more directly perceived, understandable; And, be convenient to computing machine recognition and analysis owing to converted digital form to.
Description of drawings:
Fig. 1 is the RAPD finger-print of the primer S42 of the present invention's foundation, and wherein: M is a DGL 2000DNA sign; 1 is " summer light " female parent; 2 is " summer light "; 3: be " summer light " male parent; 4 is " summer 16 early " female parent; 5 is " summer 16 early "; 6 is " summer 16 early " male parent; What arrow marked is the complementary type characteristic fragment.
The signal of Fig. 2 RAPD finger-print line segment, wherein: 1 is " summer light " female parent; 2 is " summer light "; 3: be " summer light " male parent; 4 is " summer 16 early " female parent; 5 is " summer 16 early "; 6 is " summer 16 early " male parent.
Embodiment:
Present embodiment adopts the SDS method to extract head cabbage varieties " summer light ", " summer 16 early " and parent's genomic DNA separately thereof, makes up its dna fingerprinting by two kinds of molecule labelling methods of SRAP, RAPD and is used for seed purity and identifies.Concrete operations are as follows:
1, the test material of selecting for use:
Material is a cabbage hybrid " summer light " and " summer 16 early " and parent separately thereof
2, the extraction of wild cabbage DNA and purifying:
Extract the genomic DNA of each wild cabbage material by following program:
Get flesh tissue 2g, in liquid nitrogen, pulverize, place the 50ml centrifuge tube, add 8ml extract (100mM Tris pH 8.0,50mM EDTA, 500mM NaCl, 1.5%SDS), following 30 minutes of 65 ℃ of situations, put upside down mixing frequently, add 2.5ml 5M potassium acetate ice bath 10 minutes, add the .5ml chloroform, put upside down mixing, centrifugal 8 minutes of 10000rmp gets supernatant and moves into new pipe, adds the pre-cold isopropanol of 2/3 volume, put upside down mixing, put 1-2 hour for-20 ℃, choose cotton-shaped white DNA, 70% ethanol is washed 1-2 time, dry up, be dissolved among 100ul (or an amount of) TE, and on 1% Ago-Gel electrophoresis, compare with standard λ DNA, the concentration of estimation wild cabbage DNA sample obtains high-purity wild cabbage DNA.
3, be that template is carried out the RAPD analysis with obtaining high-purity wild cabbage DNA:
RAPD is reflected on the Mastercycler 5333 type PCR instrument and finishes, and primer is S42 (worker company product is given birth in Shanghai).The RAPD-PCR reaction system is for being 20 μ l, wherein 25mmol/L MgCl
22.0 μ l; 10 * PCR Buffer, 2.0 μ l; 10mmol/L dNTP 0.5 μ l; 5U/ μ l Taq E 0.5 μ l; 20ng/ μ lPrimer 1.5 μ l; 10ng/ μ l template DNA 3 μ l; Sterilization distilled water 10.5 μ l.Suitable RAPD amplification program is: 94 ℃ of 5min; 94 ℃ of 1min, 37 ℃ of 1min, 72 ℃ of 1.5min (40 cycles); 72 ℃ of 10min.It is 15gL that concentration is adopted in the electrophoretic analysis of RAPD amplified production
-1Ago-Gel (contain 0.15ng L
-1EB), electrophoretic buffer is 1 * TAE, keeps 90V constant voltage (4～5Vcm
-1) electrophoresis 1.5～2h, gel imaging instrument imaging analysis.
4, RAPD primer screening:
Be that template DNA screens 200 RAPD random primers with " summer 16 early " kind earlier, select and be applicable to 50 of the abundant primers of wild cabbage and amplified production band, again Hybrid and its parent DNA are carried out pcr amplification respectively by two groups of materials of the order composition of female parent, cenospecies, male parent.In 50 primers, two groups of material amplification performances there are bands of a spectrum types such as weak band, Idiotype, inclined to one side supertype, inclined to one side parent form, complementary type.Can be respectively S42, S103, S193 and S42, S89, S151 to the primer that " summer light ", " summer 16 early " two groups of materials amplify the complementary type bands of a spectrum.Wherein primer S42 identifies all effective to 2 varieties, its complementary characteristic clip size that " summer light " template group amplification is produced is about 550 and 1050bp, and the complementary characteristic clip size that " summer 16 early " template group amplification is produced is about 300,600 and 875bp (Fig. 1).
5, the foundation of digital finger-print is according to above-mentioned RAPD finger-print, represent having or not of DNA band that certain allele site amplifies respectively with 1 and 0, according to from top to bottom promptly by the tape reading direction of small fragment to big fragment, the RAPD finger-print is converted to the character string of forming by 1 and 0, promptly constitutes digital finger-print.For conveniently setting up the corresponding digital fingerprint, can draw line segment synoptic diagram (Fig. 2) intuitively according to the RAPD finger-print earlier, set up digital finger-print according to synoptic diagram again, by the data that " 1 " and " 0 " are formed, promptly " summer light " maternal data are 10111111111101111; The data of " summer light " are 11111111110111; The data of " summer light " male parent are 11111111011111; " summer 16 early ", maternal data were 10111111111101111; The data in " summer 16 early " are 11111011010010001; " summer 16 early " data of male parent are 11110101111100000.Have amplified band to exist on certain position in wherein said numeral " 1 " the expression collection of illustrative plates, digital " 0 " is illustrated in does not have amplified band to exist on certain position.
Dna fingerprinting of the present invention is used for the evaluation of " summer light " and " summer 16 early " seed purity:
From " summer light " and " summer 16 early " cenospecies that production of hybrid seeds base produces, respectively randomly draw 50-80 individual plant, extract the DNA of each individual plant sample with the minim DNA extraction method, with the DNA that extracts as template, with the S42 primer it is carried out the RAPD amplification, amplified production carries out electrophoretic analysis on 1.5% agarose.If being " 11111111110111 ", the individual plant dna fingerprint that utilizes the S42 labeled analysis to find is " summer light " true hybrid strain, dna fingerprint is that " 10111100110111 " or " 11111111011111 " are " summer light " parental autocopulation strain, pseudostationary strain just.If the individual plant dna fingerprint that utilizes the S42 labeled analysis to find is " 11111011010010001 " to be " early summer 16 " true hybrid strain, dna fingerprint is that " 10111111111101111 " or " 11110101111100000 " are " summer 16 early " parental autocopulation strain, pseudostationary strain just.Use the strain number of pseudostationary strain number then, can obtain the purity of the cabbage hybrid of production of hybrid seeds base production, not only save time but also convenient, can provide purity information in time for the sale of these cabbage hybrid than field planting investigation purity divided by bulk analysis.If the purity of identifying is lower than 85%, just can not sell as seed, just may avoid pseudosperm to hinder the generation of farming part.If certified seed purity is higher than more than 95%, just can be used as the elaboration seed and sell high quality and favourable price, the economic benefit that peasant and seed dealer are all obtained.
Claims (4)
1. the method for building up of wild cabbage dna fingerprinting is characterized in that, comprises the steps:
1) extraction and the genomic DNA of purifying wild cabbage;
2) be that template is carried out the RAPD analysis with the wild cabbage DNA that obtains;
3) select representational polymorphism RAPD amplified band, having, not having and make up according to selected band for trying the material dna fingerprinting.
2. method according to claim 1 is characterized in that, cabbage hybrid " summer light ", " summer 16 early " and parent's thereof dna fingerprinting, and by the data that " 1 " and " 0 " are formed, " summer light " maternal data are 10111100110111; The data of " summer light " are 11111111110111; The data of " summer light " male parent are 11111111011111; " summer 16 early ", maternal data were 10111111111101111; The data in " summer 16 early " are 11111011010010001; " summer 16 early " data of male parent are 11110101111100000, have amplified band to exist on certain position in wherein said numeral " 1 " the expression collection of illustrative plates, and digital " 0 " is illustrated in does not have amplified band to exist on certain position.
3. method according to claim 1 is characterized in that, the primer that adopts in the described RAPD amplification is S42, and its base sequence is GGACCCAACC.
4. the application of the dna fingerprinting of the described method foundation of claim 2 is characterized in that, is used for the evaluation of cabbage hybrid " summer light " and " summer 16 early " seed purity.
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| CNA2006101182549A CN101178388A (en) | 2006-11-10 | 2006-11-10 | Establishment of cabbage hybrid Xiaguang and Zaoxia 16 fingerprint |
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| CNA2006101182549A CN101178388A (en) | 2006-11-10 | 2006-11-10 | Establishment of cabbage hybrid Xiaguang and Zaoxia 16 fingerprint |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108394A (en) * | 2009-12-25 | 2011-06-29 | 上海市农业科学院 | Method for quickly identifying seeds of seven brassica crops |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108394A (en) * | 2009-12-25 | 2011-06-29 | 上海市农业科学院 | Method for quickly identifying seeds of seven brassica crops |
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Open date: 20080514 |