CN102108394A - Method for quickly identifying seeds of seven brassica crops - Google Patents
Method for quickly identifying seeds of seven brassica crops Download PDFInfo
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- CN102108394A CN102108394A CN2009102008308A CN200910200830A CN102108394A CN 102108394 A CN102108394 A CN 102108394A CN 2009102008308 A CN2009102008308 A CN 2009102008308A CN 200910200830 A CN200910200830 A CN 200910200830A CN 102108394 A CN102108394 A CN 102108394A
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Abstract
The invention discloses a method for quickly identifying seeds of seven brassica crops, namely brassica campestris ssp.chinensis, brassica campestris ssp.pekinensis, asparagus broccoli, cauliflower, ball cabbage, turnip cabbage and brassica napus. The method comprises the following steps: 1, extracting DNA of the seeds of the seven brassica crops; 2, performing SRAP (sequence-related amplified polymorphism) and ISSR (inter-simple sequence repeat) analysis on the acquired DNA of the seven brassica crops as templates; and 3, selecting representative polymorphic amplified bands for constructing DNA finger prints of the tested materials, wherein all the seven brassica crops have specific DNA fingerprints in the DNA finger prints, thus distinguishing the seven brassica crops. The seed identification method disclosed by the invention is represented in a form of DNA finger prints which are visual and clear, and the DNA finger prints are converted into a digital representing form convenient for recognition and analysis of a computer. By the method disclosed by the invention, the seeds of the seven brassica crops with near genetic relationships and high resemblance of the shapes of the seeds can be identified on inheritance, the results are accurate and reliable, and the detection is quick.
Description
Technical field
The present invention relates to rape and belong to the crop seed authenticate technology, specifically use the dna molecular marker technology and differentiate that fast seven kinds of rapes belong to the method for crop seed.
Background technology
It is various that rape belongs to crop species, wherein cultivated area is bigger crops such as Chinese cabbage, Chinese cabbage, broccoli, Cauliflower, cabbage, kohlrabi and swede type rape, sibship is near between them, shape of the seed similarity degree height, be difficult to they are distinguished fast with ordinary method, and molecular marking technique such as RAPD, SRAP, ISSR etc., by the amplification bands of a spectrum between the comparative material, just can not only save time but also reliable preferably with its differentiation.SRAP (sequence-related amplifiedpolymorphism) is based on a kind of novel molecular labeling technique of PCR, by California, USA university vegetable crop is that Li and doctor Quiros developed at first in calendar year 2001, this technology integrates RAPD and AFLP two technological merits, have simple, characteristics such as stablize, in genome, be evenly distributed, now be used for the research of plant such as potato, apple, rape, cotton and rice blast.ISSR (inter-simple sequence repeat) claims again to increase between the simple sequence iteron, it is simple repeated sequence (SSR) the design primer that utilizes the eukaryotic gene group extensively to exist, need not to predict genome sequence, all crops all are suitable for, and experimental cost is low, simple to operate, good stability, rich polymorphism, now has been widely used in research fields such as the assignment of genes gene mapping, seed resource classification, biological heredity diversity analysis and genetic map construction.Therefore, utilize molecular marking technique to develop the method that seven kinds of rapes of a kind of quick discriminating belong to crop seed, can before commodity seed is sold, carry out purity and identify, hinder the generation of farming part to avoid pseudosperm.
Summary of the invention
The purpose of this invention is to provide the method that seven kinds of rapes of a kind of quick discriminating belong to crop seed, to overcome the deficiency that prior art exists.
Technical conceive of the present invention is such:
The dna fingerprinting identification method is the molecule marking method based on the polymorphism of DNA, can remedy and overcome many defectives and a difficult problem in the morphology evaluation of seed and isozyme, seed protein electrophoresis are identified.Seven kinds of rapes of the present invention belong to the dna fingerprinting of crop, the data of being made up of " 1 " and " 0 ".Use primer M9-GA18, the data of Chinese cabbage, Chinese cabbage, broccoli, Cauliflower, cabbage, kohlrabi and swede type rape are respectively 01111101011101111000101000101,10101101011101101000101010101,10010110110100010101010001010,10010100110110000110010001010,10010100110111110111010101100,10010100110110000110010000010 and 10011101011110011100011000011.Use the data of primer UBC810 to be respectively: 10000110100101000110000,10001101101101001010000,00000001011000010101010,00110001010010010101011,01110000100110100001011,00110001010000010101011 and 00000000000101110110100.Use the data of primer UBC834 to be respectively: 1100101001101101001,1100100100000000100,0110100001001000011,1110101000010110011,1110111001010110011,1110001000010110011 and 0001101011111100011.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 Chinese cabbage, Chinese cabbage, broccoli, Cauliflower, cabbage, kohlrabi, swede type rape can adopt the product of Inst. of Gardening, Academy of Agriculture of Shanghai's public offering.
Seven kinds of rapes belong to the method that crop seed is differentiated fast, comprise the steps:
1) genomic dna that, belongs to crop with seed extraction and seven kinds of rapes of purifying;
2), belonging to crop DNA with seven kinds of rapes that obtain is that template is carried out SRAP and ISSR analyzes;
3), select representational polymorphism amplified band, having, not having and make up according to selected band for trying the material dna fingerprinting.
Wherein said seven kinds of rapes belong to crop all its special dna fingerprint, can make a distinction mutually.The primer that adopts in described SRAP, the ISSR amplification is to give birth to worker's biotechnology company limited synthetic Oligonucleolide primers by Shanghai.The primer that the SRAP amplification is adopted is M9:TGAGTCCAAACCGGTAG, GA18:GGCTTGAACGAGTGACTGA.The primer that the ISSR amplification is adopted is UBC810:GAGAGAGAGAGAGAGAT, UBC834:AGAGAGAGAGAGAGAGYT.
Method of the present invention can be applied to the quick discriminating of totally seven kinds of rape genus crop seeds of Chinese cabbage, Chinese cabbage, broccoli, Cauliflower, cabbage, kohlrabi and swede type rape.
The present invention has following advantage:
1, the present invention has created the method that seven kinds of rapes of a kind of quick discriminating belong to crop seed, and this method can be identified seven kinds of rapes genus crop seeds from inheritance, and is therefore accurate, reliable.
2, adopt the present invention, when seven kinds of rapes genus crop seeds are detected, with present widespread use fast, the minim DNA extraction method extracts the DNA of seed sample to be measured, with the DNA that extracts as template, carrying out SRAP or ISSR analyzes, in several hrs, just can know for the dna fingerprint of examination material, therefore, just can judge its verity rapidly.
3, because seed discrimination method of the present invention is to represent with the form of dna fingerprinting, seem more directly perceived, understandable; And, be convenient to computer recognition and analysis owing to converted digital form to.
Description of drawings:
SRAP, ISSR finger printing that accompanying drawing is set up for the present invention, wherein: M is DGL 2000DNA sign; 1,2,3,4,5,6,7: represent Chinese cabbage, Chinese cabbage, broccoli, Cauliflower, cabbage, kohlrabi and swede type rape respectively.A, B, C: the amplification electrophorogram that is followed successively by primer M9-GA18, UBC810 and UBC834.
Embodiment:
Present embodiment adopts the CTAB method to extract the genomic dna that seven kinds of rapes belong to crop seed, makes up the discriminating that its dna fingerprinting is used for seed by two kinds of molecule marking methods of SRAP, ISSR.Concrete operations are as follows:
1, the test materials of selecting for use:
Material is the seed of Chinese cabbage, Chinese cabbage, broccoli, Cauliflower, cabbage, kohlrabi and swede type rape.
2, the extraction of seed DNA and purifying:
Extract the genomic dna of each part seed material by following program:
Get 1 or 0.1-0.2g seed; place the mortar that falls excess temperature through-20 ℃ of refrigerators to grind; change in the centrifuge tube of 1.5mL; 1 * DNA extraction liquid (the 50mmol/LTris-HCl pH7.5 that adds 55 ℃ of preheatings of 0.35mL; 10mmol/LEDTApH8.0; 0.7mmol/L NaCl; 1% beta-mercaptoethanol; 1%CTAB); add 2 * DNA extraction liquid (concentration is the former twice) behind the 3min; putting into 55 ℃ of incubators spends the night; put upside down therebetween for several times; add the saturated phenol of 0.7mL; go up mixing extracting 3h at homemade vortex mixer (2r/min); centrifugal 10min under the 6000r/min; supernatant liquor is changed over to another pipe; add the saturated phenol of 0.35mL and 0.35mL chloroform-primary isoamyl alcohol (24: 1) extracting 2h; centrifugal 10min under the 6000r/min; get supernatant liquor to another pipe, add 0.7mL chloroform-primary isoamyl alcohol (24: 1) extracting 2h, centrifugal 10min under the 6000r/min; get supernatant liquor to another pipe; add 0.07mL3mol/LNaAc and 0.7mL Virahol, precipitate 30min under the room temperature, centrifugal 5min under the 3000r/min; abandon supernatant liquor; with 70% alcohol washing DNA, dry up, be dissolved among 100 μ L (or an amount of) TE; and on 1% sepharose electrophoresis; compare with standard λ DNA, the concentration of estimation DNA sample obtains high purity DNA.
3, be that template is carried out SRAP, ISSR analysis with obtaining DNA:
The SRAP amplified reaction adopts the reaction system of 20 μ L, 25mmol/L MgCl2 2.0 μ L wherein, 10 * PCR Buffer, 2.0 μ L, 10mmol/L dNTP 0.4 μ L, 5U/ μ L Taq archaeal dna polymerase 0.2 μ L, each 3.0 μ L of 0.1 μ mol/L primer, 10ng/ μ L template DNA 3.0 μ L, sterilization distilled water 6.4 μ L add a cover a mineral oil and increase.Amplification program is 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 35 ℃ of annealing 1min, 72 ℃ are extended 1.5min, 5 circulations; 94 ℃ of sex change 1min, 50 ℃ of annealing 1min, 72 ℃ are extended 1.5min, 35 circulations; 4 ℃ of preservations behind 72 ℃ of extension 10min.Amplified production is with carrying out electrophoretic analysis on 1.5% sepharose.After reaction finished, the PCR product used 6% polyacrylamide gel electrophoresis 1.5 hours, took pictures after adopting quick argentation dyeing photographic fixing.
The ISSR amplified reaction adopts the reaction system of 20 μ L, 25mmol/L MgCl2 2.0 μ L wherein, 10 * PCR Buffer, 2.0 μ L, 10mmol/L dNTP 0.4 μ L, 5U/ μ L Taq archaeal dna polymerase 0.2 μ L, 0.1 μ mol/L primer, 3.0 μ L, 10ng/ μ L template DNA 3.0 μ L, sterilization distilled water 9.4 μ L add a cover a mineral oil and increase.Amplification program is 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 52 ℃ of annealing 1min, 72 ℃ are extended 1.5min, 35 circulations; 4 ℃ of preservations behind 72 ℃ of extension 10min.Amplified production is with carrying out electrophoretic analysis on 1.5% sepharose.The PCR product is electrophoresis 1.5h in 1.5% sepharose, EB dyeing, and Gel DocTM EQ 170-8060 gel imaging instrument is observed and photographic analysis.
4, primer screening:
Use 100 ISSR primers and 35 pairs of SRAP primers that seven parts of materials are carried out pcr amplification, wherein there are 44 ISSR primers and 32 pairs of SRAP primers can amplify band clearly, but the amplification of most of primers can not belong to crop with 7 portions of rapes to be distinguished significantly, can not play the effect of discriminating.Finishing screen is selected 2 ISSR primers (UBC810 and UBC834) and 1 pair of SRAP primer (M9-GA18) can amplify good polymorphic bands, 7 portions of rapes can be belonged to crop and make a distinction one by one.
5, the foundation of digital finger-print:
According to above-mentioned two kinds of molecular marking fingerprints, represent having or not of certain allelotrope site DNA amplification band respectively with 1 and 0, according to from top to bottom promptly by the tape reading direction of big fragment to small segment, molecular marking fingerprint is converted to the character string of forming by 1 and 0, promptly constitute digital finger-print, the data of forming by " 1 " and " 0 ".Use primer M9-GA18, the data of Chinese cabbage, Chinese cabbage, broccoli, Cauliflower, cabbage, kohlrabi and swede type rape are respectively 01111101011101111000101000101,10101101011101101000101010101,10010110110100010101010001010,10010100110110000110010001010,10010100110111110111010101100,10010100110110000110010000010 and 10011101011110011100011000011.Use the data of primer UBC810 to be respectively: 10000110100101000110000,10001101101101001010000,00000001011000010101010,00110001010010010101011,01110000100110100001011,00110001010000010101011 and 00000000000101110110100.Use the data of primer UBC834 to be respectively: 1100101001101101001,1100100100000000100,0110100001001000011,1110101000010110011,1110111001010110011,1110001000010110011 and 0001101011111100011.
Dna fingerprinting of the present invention can be used for the quick discriminating that rape belongs to vegetable seed:
The rape that produces from production of hybrid seeds base belongs to the vegetable seed respectively randomly draws the 50-80 grain, and the DNA with minim DNA extraction method extraction seed as template, carries out pcr amplification and electrophoretic analysis with corresponding primer to it with the DNA that extracts.As the seed dna fingerprint more than 95% that utilizes primer UBC834 analysis to find is that " 1100101001101101001 " are Chinese cabbage (green vegetables) kind, dna fingerprint is that " 1100100100000000100 " are Chinese cabbage (Chinese cabbage) kind, dna fingerprint is that " 0110100001001000011 " is the broccoli kind, dna fingerprint is that " 1110101000010110011 " are that " 1110111001010110011 " are the cabbage kind for cauliflower variety, dna fingerprint, and dna fingerprint is that " 1110001000010110011 " are the kohlrabi kind.If the purity of identifying is lower than 90%, just can not sell as seed, hinder the generation of farming part to avoid pseudosperm.If have the dna fingerprint of " 0001101011111100011 " in the above vegetable variety seed, just the swede type rape seed is sneaked in explanation, if the ratio of sneaking into is higher, just might be deliberately to sneak into or mechanical admixture causes, and such seed must be scrapped.If sneak in the vegetable seed with ablastous Semen Brassicae campestris, though can not occur assorted strain behind the seed sprouting, but can reduce seed germination rate, it also is underproof seed, can identify rape with method of the present invention and belong to the ratio of sneaking into the swede type rape seed in the vegetable variety, can disclose illegal retailer serves as the rape genus vegetable seed of high value with the similar cheap Semen Brassicae campestris of shape of the seed way.
Claims (4)
1. differentiate that fast seven kinds of rapes belong to the method for crop seed for one kind, it is characterized in that, comprise the steps:
1) DNA that, belongs to the crop gene group with seed extraction and seven kinds of rapes of purifying;
2), belonging to crop DNA with seven kinds of rapes that obtain is that template is carried out SRAP and ISSR analyzes;
3), select representational polymorphism 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, seven kinds of rapes belong to the dna fingerprinting of crops, the data of being made up of " 1 " and " 0 ".Use primer M9-GA18, the data of Chinese cabbage, Chinese cabbage, broccoli, Cauliflower, cabbage, kohlrabi and swede type rape are respectively 01111101011101111000101000101,10101101011101101000101010101,10010110110100010101010001010,10010100110110000110010001010,10010100110111110111010101100,10010100110110000110010000010 and 10011101011110011100011000011.Use the data of primer UBC810 to be respectively: 10000110100101000110000,10001101101101001010000,00000001011000010101010,00110001010010010101011,01110000100110100001011,00110001010000010101011 and 00000000000101110110100.Use the data of primer UBC834 to be respectively: 1100101001101101001,1100100100000000100,0110100001001000011,1110101000010110011,1110111001010110011,1110001000010110011 and 0001101011111100011.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.
3. method according to claim 1 is characterized in that, the primer that adopts in the described SRAP amplification is M9:TGAGTCCAAACCGGTAG, GA18:GGCTTGAACGAGTGACTGA.The primer that adopts in the ISSR amplification is UBC810:GAGAGAGAGAGAGAGAT, UBC834:AGAGAGAGAGAGAGAGYT.
4. the application of the described method of claim 2 is characterized in that, it is near to be used for sibship, and seven kinds of rapes that the shape of the seed similarity degree is high belong to the quick discriminating of crop seed.
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| CN2009102008308A CN102108394A (en) | 2009-12-25 | 2009-12-25 | Method for quickly identifying seeds of seven brassica crops |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703598A (en) * | 2012-07-04 | 2012-10-03 | 中国热带农业科学院香料饮料研究所 | Method for identifying pepper germplasm resources |
| CN110804675A (en) * | 2019-11-21 | 2020-02-18 | 南京农业大学 | Microsatellite DNA marker fingerprint spectrum of non-heading Chinese cabbage and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101178388A (en) * | 2006-11-10 | 2008-05-14 | 上海市农业科学院 | Establishment of cabbage hybrid Xiaguang and Zaoxia 16 fingerprint |
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- 2009-12-25 CN CN2009102008308A patent/CN102108394A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101178388A (en) * | 2006-11-10 | 2008-05-14 | 上海市农业科学院 | Establishment of cabbage hybrid Xiaguang and Zaoxia 16 fingerprint |
Non-Patent Citations (2)
| Title |
|---|
| 马金骏: "不结球白菜种质资源遗传多样性的初步研究", 《中国优秀硕士学位论文全文数据库（农业科技辑）》 * |
| 马金骏等: "RAPD、ISSR、SRAP技术构建不结球白菜杂交种指纹图谱", 《中国园艺学会十字花科蔬菜分会第六届研讨会暨新品种展示会论文集》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703598A (en) * | 2012-07-04 | 2012-10-03 | 中国热带农业科学院香料饮料研究所 | Method for identifying pepper germplasm resources |
| CN102703598B (en) * | 2012-07-04 | 2014-01-15 | 中国热带农业科学院香料饮料研究所 | Method for identifying pepper germplasm resources |
| CN110804675A (en) * | 2019-11-21 | 2020-02-18 | 南京农业大学 | Microsatellite DNA marker fingerprint spectrum of non-heading Chinese cabbage and application thereof |
| CN110804675B (en) * | 2019-11-21 | 2022-07-22 | 南京农业大学 | Microsatellite DNA marker fingerprint spectrum of non-heading Chinese cabbage and application thereof |
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