CN111549174A - Special primer for identifying variety of water chestnut and water chestnut variety identification method - Google Patents
Special primer for identifying variety of water chestnut and water chestnut variety identification method Download PDFInfo
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- CN111549174A CN111549174A CN202010630352.0A CN202010630352A CN111549174A CN 111549174 A CN111549174 A CN 111549174A CN 202010630352 A CN202010630352 A CN 202010630352A CN 111549174 A CN111549174 A CN 111549174A
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- 235000003283 Pachira macrocarpa Nutrition 0.000 title claims abstract description 41
- 235000014364 Trapa natans Nutrition 0.000 title claims abstract description 41
- 235000009165 saligot Nutrition 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 20
- 241001083492 Trapa Species 0.000 title claims description 35
- 210000000003 hoof Anatomy 0.000 claims abstract description 30
- 238000012408 PCR amplification Methods 0.000 claims abstract description 7
- 240000001085 Trapa natans Species 0.000 claims abstract description 6
- 239000000499 gel Substances 0.000 claims description 9
- 239000011543 agarose gel Substances 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 6
- 238000001962 electrophoresis Methods 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 108010006785 Taq Polymerase Proteins 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 239000007850 fluorescent dye Substances 0.000 claims description 3
- JGBUYEVOKHLFID-UHFFFAOYSA-N gelred Chemical compound [I-].[I-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCCCC(=O)NCCCOCCOCCOCCCNC(=O)CCCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 JGBUYEVOKHLFID-UHFFFAOYSA-N 0.000 claims description 3
- 150000007523 nucleic acids Chemical class 0.000 claims description 3
- 102000039446 nucleic acids Human genes 0.000 claims description 3
- 108020004707 nucleic acids Proteins 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000012154 double-distilled water Substances 0.000 claims description 2
- 238000012257 pre-denaturation Methods 0.000 claims description 2
- 241000894007 species Species 0.000 abstract description 11
- 230000008569 process Effects 0.000 abstract description 2
- 244000285774 Cyperus esculentus Species 0.000 abstract 2
- 235000005853 Cyperus esculentus Nutrition 0.000 abstract 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241000209117 Panicum Species 0.000 description 1
- 235000006443 Panicum miliaceum subsp. miliaceum Nutrition 0.000 description 1
- 235000009037 Panicum miliaceum subsp. ruderale Nutrition 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a primer special for identifying chufa varieties and a chufa variety identification method thereof, belonging to the field of variety identification and having a primer sequence of AGAGAGAGAGAGAGAGYC. By utilizing ISSR marking technology to synthesize primers, carrying out PCR amplification on local species of the wine, namely Gui hoof No. 2 and Gui hoof No. 3, the repeatability is good, the local species ratio of the Gui hoof No. 3 to the wine is that the Gui hoof No. 3 is deleted at about 730bp, the genome of the Gui hoof No. 3 is present at about 250bp, and the genome of the Gui hoof No. 2 is deleted. The fact that the local species of the Guizhou No. 3 and the Panyu water chestnut and the Guizhou No. 2 have obvious difference in ISSR marking technology proves that the primers can identify the 3 varieties; the operation is simple, convenient and quick, the sample is easy to process, and the identification result is highly accurate.
Description
Technical Field
The invention relates to the field of variety identification, in particular to a primer special for identifying water chestnut varieties and a water chestnut variety identification method.
Background
The water chestnuts are commonly called as water chestnuts, can be eaten fresh or processed, are rich in nutrition, are one of famous and special-quality agricultural products in Guangxi and important export-induced products, and are listed as special agricultural products for the important development of agriculture in China.
At present, Guangxi is the first large-yield area of water chestnuts in China, and the planting area accounts for more than 50% of the national planting area. But is influenced by the factors of single variety, degeneration of species, resistance, low fruit bearing rate and the like of the water chestnuts, and the industrial development is greatly limited. Corresponding standardized cultivation techniques cannot be designed according to specific varieties, and good breeding of improved varieties cannot be ensured.
Disclosure of Invention
The invention aims to provide a primer special for identifying a water chestnut variety and a water chestnut variety identification method thereof, and solves the technical problems mentioned in the background technology.
A primer special for identifying water chestnut varieties has a sequence of AGAGAGAGAGAGAGAGYC.
Further, the water chestnut varieties comprise local species of the Panum Panicum, Guizhou No. 2 and Guizhou No. 3.
A water chestnut variety identification method of a primer special for water chestnut variety identification comprises the following steps:
step 1: establishing an ISSR amplification system of PCR and an ISSR reaction program;
step 2: extracting DNA of water chestnut varieties to be detected, wherein the water chestnut varieties comprise local varieties of Panyu with wine, Gui hoof No. 2 and Gui hoof No. 3;
and step 3: using the DNA extracted by the special primer pair in the step 1 as a template to carry out PCR amplification;
and 4, step 4: preparing 1.5% agarose gel containing 0.01% nucleic acid gel fluorescent dye GelRed (10000x), taking PCR amplification products to carry out electrophoresis in 1 xTBE buffer solution, taking out the gel after bromophenol blue migrates to 2/3 of the agarose gel, rinsing with clear water, and placing in an automatic gel imager for observation and photographing for recording.
Further, the 20.0. mu.l ISSR amplification system in step 1 included 14.7. mu.l ddH2O、2.0μl10×ExTaq buffer、1.6. mu.l dNTP, 0.5. mu.l Primer, 0.2. mu.l Ex Taq DNA polymerase and 1.0. mu.l genomicDNA.
Further, the dNTP was 2.5mmol/L, the Primer was 10. mu. mol/L, the Ex Taq DNApolymerase was 5U/. mu.l, and the Genomic DNA was 80 ng/. mu.l.
Further, the ISSR reaction procedure in step 1 comprises pre-denaturation at 94 ℃ for 4 min; 40 cycles, 94 ℃ 30 sec; 40 cycles, 50-54 ℃ for 40 sec; 40 cycles, 72 ℃ 90 sec; 7min at 72 ℃; finally, the product is stored at 4 ℃.
Further, in the step 4, 7. mu.l of the amplified product was electrophoresed in 1 XTBE buffer at a voltage of 5 v/cm.
By adopting the technical scheme, the invention has the following technical effects:
the invention synthesizes a primer (AGAGAGAGAGAGAGAGYC) by utilizing an ISSR marking technology, PCR amplification is carried out on local seeds of the wine, namely, Gui hoof No. 2 and Gui hoof No. 3, the repeatability is good, the Gui hoof No. 3 has a deletion of about 730bp compared with the local seeds of the wine, the Gui hoof No. 3 has a genome of about 250bp compared with the Gui hoof No. 2, and the Gui hoof No. 2 has a deletion. Proved that the local species of the water chestnut of Guizhou No. 3 and the water chestnut of Panyu with the Guizhou No. 2 have obvious difference in ISSR marking technology, which indicates that the primer (AGAGAGAGAGAGAGAGYC) can identify the above 3 varieties; the operation is simple, convenient and quick, the sample is easy to process, and the identification result is highly accurate.
Drawings
FIG. 1 is a molecular signature of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings by way of examples of preferred embodiments. It should be noted, however, that the numerous details set forth in the description are merely for the purpose of providing the reader with a thorough understanding of one or more aspects of the present invention, which may be practiced without these specific details.
A primer special for identifying water chestnut varieties has a sequence of AGAGAGAGAGAGAGAGYC. The water chestnut varieties used for identification by the primers comprise local species of Panyu, Guizhou No. 2 and Guizhou No. 3.
A water chestnut variety identification method of a primer special for water chestnut variety identification comprises the following steps:
establishment of ISSR technical system
The following ISSR amplification system was established:
note: the reaction solution for PCR was prepared in an ice bath and then placed on a PCR instrument for PCR reaction.
Second, ISSR reaction procedure
The improved reaction procedure was:
third, electrophoresis detection
Preparing 1.5% agarose gel containing 0.01% nucleic acid gel fluorescent dye GelRed (10000x), taking 7 mul of amplification product, carrying out electrophoresis in 1 xTBE buffer solution at the electrophoresis voltage of 5v/cm, taking out the gel after bromophenol blue migrates to 2/3 parts of the agarose gel, rinsing with clear water, and placing in an automatic gel imager for observation and photographing for recording.
Fourthly, obtaining a result:
a primer (AGAGAGAGAGAGAGAGYC) is synthesized by ISSR marking technology, PCR amplification is carried out on local species of the Panyu wine, Gui hoof No. 2 and Gui hoof No. 3, and the repeatability is good, as shown in figure 1. The species ratio of Gui hoof No. 3 to Panyu wine is that Gui hoof No. 3 is deleted at about 730bp, compared with Gui hoof No. 2, Gui hoof No. 3 exists at about 250bp of genome, and Gui hoof No. 2 is deleted. The fact that the local species of the Guizhou No. 3 and the Panyu water chestnut and the Guizhou No. 2 are obviously different in ISSR marking technology is proved. The introduction primer (AGAGAGAGAGAGAGAGYC) can identify the above 3 varieties. In FIG. 1, the symbol M is DL 2000 standard molecular weight; 1 is local seed of edible black wine; 2 is Gui Zu No. 2; 3 is Gui Zu No. 3. The positions pointed by the two arrows illustrate: the species ratio of Gui hoof No. 3 to Panyu wine is that Gui hoof No. 3 is deleted at about 730bp, compared with Gui hoof No. 2, Gui hoof No. 3 exists at about 250bp of genome, and Gui hoof No. 2 is deleted.
2000bp to the left, 1500 bp: the marker M is DL 2000 standard molecular weight, 250bp, 500bp, 750bp, 1500bp and 2000bp are used as standards, and are references of right 1, 2 and 3.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.
Claims (7)
1. A primer special for identifying water chestnut varieties is characterized by comprising the following steps: the primer sequence is AGAGAGAGAGAGAGAGYC.
2. The primer special for identifying the variety of the water chestnuts as claimed in claim 1, wherein the primer is characterized in that: the water chestnut varieties comprise local seeds of Panyu, Gui hoof No. 2 and Gui hoof No. 3.
3. The water chestnut variety identification method of the primer special for water chestnut variety identification according to claim 1, characterized in that: the method comprises the following steps:
step 1: establishing an ISSR amplification system of PCR and an ISSR reaction program;
step 2: extracting DNA of water chestnut varieties to be detected, wherein the water chestnut varieties comprise local varieties of Panyu with wine, Gui hoof No. 2 and Gui hoof No. 3;
and step 3: using the DNA extracted by the special primer pair in the step 1 as a template to carry out PCR amplification;
and 4, step 4: preparing 1.5% agarose gel containing 0.01% nucleic acid gel fluorescent dye GelRed (10000x), taking PCR amplification products to carry out electrophoresis in 1 xTBE buffer solution, taking out the gel after bromophenol blue migrates to 2/3 of the agarose gel, rinsing with clear water, and placing in an automatic gel imager for observation and photographing for recording.
4. The water chestnut variety identification method of the primer special for water chestnut variety identification according to claim 3, characterized in that: the 20.0. mu.l ISSR amplification system in step 1 included 14.7. mu.l ddH2O, 2.0. mu.l 10 × Ex Taq buffer, 1.6. mu.l dNTP, 0.5. mu.l Primer, 0.2. mu.l Ex Taq DNA polymerase and 1.0. mu.l Genomic DNA.
5. The water chestnut variety identification method of the primer special for water chestnut variety identification according to claim 4, characterized in that: the dNTP is 2.5mmol/L, the Primer is 10 mu mol/L, the Ex Taq DNA polymerase is 5U/mu L, and the Genomic DNA is 80 ng/mu L.
6. The water chestnut variety identification method of the primer special for water chestnut variety identification according to claim 5, characterized in that: the ISSR reaction procedure in the step 1 comprises pre-denaturation at 94 ℃ for 4 min; 40 cycles, 94 ℃ 30 sec; 40 cycles, 50-54 ℃ for 40 sec; 40 cycles, 72 ℃ 90 sec; 7min at 72 ℃; finally, the product is stored at 4 ℃.
7. The water chestnut variety identification method of the primer special for water chestnut variety identification according to claim 5, characterized in that: in the step 4, 7 μ l of the amplified product is electrophoresed in 1 XTBE buffer solution, and the voltage for electrophoresis is 5 v/cm.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531844A (en) * | 2014-12-05 | 2015-04-22 | 中国农业科学院郑州果树研究所 | SSR genotype-based fruit tree variety distinguishing and characteristic fingerprint displaying method |
| CN106967814A (en) * | 2017-04-19 | 2017-07-21 | 浙江海洋大学 | Differentiate the method for the naked skin-carp meat in Xinjiang of south and north Sinkiang distribution using ISSR molecular marking techniques |
| CN107541506A (en) * | 2016-06-23 | 2018-01-05 | 新疆农业大学 | A kind of Xinjiang garlic wild relatives DNA extracting method |
| CN109207620A (en) * | 2018-09-30 | 2019-01-15 | 江苏省中国科学院植物研究所 | A kind of precious magnificent carpinus turczaninowii ISSR-PCR molecular labeling system |
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2020
- 2020-07-02 CN CN202010630352.0A patent/CN111549174B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531844A (en) * | 2014-12-05 | 2015-04-22 | 中国农业科学院郑州果树研究所 | SSR genotype-based fruit tree variety distinguishing and characteristic fingerprint displaying method |
| CN107541506A (en) * | 2016-06-23 | 2018-01-05 | 新疆农业大学 | A kind of Xinjiang garlic wild relatives DNA extracting method |
| CN106967814A (en) * | 2017-04-19 | 2017-07-21 | 浙江海洋大学 | Differentiate the method for the naked skin-carp meat in Xinjiang of south and north Sinkiang distribution using ISSR molecular marking techniques |
| CN109207620A (en) * | 2018-09-30 | 2019-01-15 | 江苏省中国科学院植物研究所 | A kind of precious magnificent carpinus turczaninowii ISSR-PCR molecular labeling system |
Non-Patent Citations (4)
| Title |
|---|
| AMNON LEVI等: "ISSR and AFLP markers differ among American watermelon cultivars with limited genetic diversity", 《J.AMER SOC HORT SCI》 * |
| 江文等: "24个荸荠品种遗传多样性RAPD分析", 《南方农业学报》 * |
| 江文等: "荸荠种质资源品种遗传多样性ISSR分析", 《西南农业学报》 * |
| 郭郁频等: "早熟禾种质资源ISSR遗传多样性分析", 《中国草地学报》 * |
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