CN116622892A - Molecular marker primer and kit for identifying rubber tree cloud-ground 3042 and application - Google Patents
Molecular marker primer and kit for identifying rubber tree cloud-ground 3042 and application Download PDFInfo
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- CN116622892A CN116622892A CN202310663966.2A CN202310663966A CN116622892A CN 116622892 A CN116622892 A CN 116622892A CN 202310663966 A CN202310663966 A CN 202310663966A CN 116622892 A CN116622892 A CN 116622892A
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- 244000043261 Hevea brasiliensis Species 0.000 title claims abstract description 49
- 239000003147 molecular marker Substances 0.000 title claims abstract description 31
- 238000011160 research Methods 0.000 claims abstract description 23
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 14
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims abstract description 6
- 238000012163 sequencing technique Methods 0.000 abstract description 8
- 238000012217 deletion Methods 0.000 abstract description 3
- 230000037430 deletion Effects 0.000 abstract description 3
- 238000003780 insertion Methods 0.000 abstract description 3
- 230000037431 insertion Effects 0.000 abstract description 3
- 238000006467 substitution reaction Methods 0.000 abstract description 3
- 239000003550 marker Substances 0.000 abstract 1
- 229920003052 natural elastomer Polymers 0.000 description 5
- 229920001194 natural rubber Polymers 0.000 description 5
- 230000003321 amplification Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 238000000227 grinding Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 238000013475 authorization Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 229920001971 elastomer Polymers 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000026487 Triploidy Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001112 coagulating effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012214 genetic breeding Methods 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 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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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- Biotechnology (AREA)
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Abstract
The invention discloses a molecular marker primer, a kit and application for identifying rubber tree cloud research 3042. The molecular marker primer comprises an upstream primer and a downstream primer, wherein the upstream primer is shown as SEQ ID NO:1, wherein the downstream primer is shown as SEQ ID NO: 2. The kit comprises the molecular marker primer. The application comprises the following steps: the molecular marker primer is used for identifying the rubber tree cloud research 3042. The identification of the cloud research 3042 is realized by utilizing the sequencing result of the marker to distinguish varieties, such as the difference of one or more bases, substitution, insertion, deletion, repetition and the like among different sequences, and the varieties of the rubber trees can be accurately identified by distinguishing the IAN873, the GT1, the cloud research 774 and the hot reclamation 516.
Description
Technical Field
The present disclosure relates to the fields of molecular biology and genetic breeding, and in particular relates to a molecular marker primer, a kit and an application for identifying rubber tree cloud research 3042.
Background
Rubber tree is a typical tree species with rainforest generated for many years, is native to Brazil, venezuela, and Guinea in America river basin, and is introduced into China later, and is mainly planted in Hainan, yunnan, guangdong and other places in China. Natural rubber is produced by coagulating and drying a latex discharged during tapping of a rubber tree. In recent years, the natural rubber demand in China is continuously increased, and the supply and demand gap is continuously increased. According to the data of the industrial information network, the natural rubber yield of 2021 in China is 85.1 tons, the demand is 594.9 tons, and the self-supporting rate is only 14.30 percent.
In the current international situation, supply safety faces a number of challenges, and domestic production needs to exert the function of ballast stone for a long time. The natural rubber has unique excellent properties in the fields of weapon equipment, aircraft tires, high-elastic shock absorption, high-altitude detection and the like, and has wide development prospect in the future natural rubber tree industry.
At present, the direct object of identification, authorization and breeding of rubber tree varieties is the character, and the character test is still an industry standard for the specificity test of a plurality of plant varieties. However, the behavior of the trait is susceptible to environmental and human factors and requires years for accurate identification. The rubber tree germplasm resources, important seed selection materials and varieties popularized and applied in production have the phenomena of homonymous foreign matters and homonymous foreign matters, which is unfavorable for the seed selection, authorization, quick popularization and intellectual property protection of the rubber tree varieties. Especially, uncertainty of the rubber tree variety in actual production can severely limit the healthy and sustainable development of the rubber industry, and the economical benefits of rubber farmers can be seriously influenced by the impure or even erroneous rubber tree seedling variety.
BRIEF SUMMARY OF THE PRESENT DISCLOSURE
In order to solve the problem of inaccurate identification of the rubber tree cloud-research 3042, the embodiment of the disclosure provides a molecular marker primer, a kit and application for identification of the rubber tree cloud-research 3042. The technical scheme is as follows:
in one aspect, the disclosure provides a molecular marker primer identified by rubber tree cloud research 3042, wherein the molecular marker primer comprises an upstream primer and a downstream primer, and the upstream primer is shown as SEQ ID NO in a sequence table: 1, wherein the downstream primer is shown as SEQ ID NO: 2.
In another aspect, the present disclosure provides a kit for identification of rubber tree cloud research 3042, the kit comprising the above-described molecular marker primer.
In yet another aspect, the present disclosure provides the use of a molecular marker primer identified by rubber tree cloud research 3042, the use comprising: the molecular marker primer is used for identifying the rubber tree cloud research 3042, and comprises an upstream primer and a downstream primer, wherein the upstream primer is shown as SEQ ID NO in a sequence table: 1, wherein the downstream primer is shown as SEQ ID NO: 2.
Further, the application further includes: the molecular marker primer can distinguish rubber tree IAN873, GT1, cloud-ground 774 and hot reclamation 516.
The technical scheme provided by the embodiment of the disclosure has the beneficial effects that: the embodiment of the invention provides a molecular marker primer for identifying the rubber tree cloud research 3042, a kit and application thereof, wherein the molecular marker primer is used for distinguishing varieties through an amplification product sequencing result, such as the difference of one or more bases, substitution, insertion, deletion, repetition and the like among different sequences, so that the identification of the rubber tree cloud research 3042 is realized, the identification results are not influenced by environment and other human factors, and the identification of the rubber tree varieties is accurately carried out.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and other drawings may be obtained according to these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is a comparison of sequences designated a-e provided in example three of the present disclosure.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present disclosure more apparent, the embodiments of the present disclosure will be described in further detail below.
Example 1
The present disclosure provides a molecular marker primer identified by rubber tree cloud research 3042, which comprises an upstream primer and a downstream primer, wherein the upstream primer is shown as SEQ ID NO:1, the downstream primer is shown as SEQ ID NO: 2. Specifically, the sequence of the upstream primer is: ATATGTCAAAATGGCTCAACAGGAG, the sequence of the downstream primer is: GCTTAGAGCAGGACATACAGAAGTA.
Example two
The present disclosure provides a kit for identifying rubber tree cloud research 3042, which comprises the molecular marker primer provided in the first embodiment.
Example III
The present disclosure provides an application of a molecular marker primer identified by rubber tree cloud research 3042, which comprises: the molecular marker primer provided in the first embodiment is used for identifying the rubber tree cloud 3042.
Further, the application further comprises: the molecular marker primer can distinguish rubber tree IAN873, GT1, cloud-ground 774 and hot reclamation 516.
Specifically, 42 rubber tree varieties are used as samples to be tested, and the variety names are shown in Table 1.
Table 1 is the sample variety name to be tested
The DNA of the above 42 samples to be tested was extracted. When the method is realized, the DNA of the sample to be detected is extracted by adopting a novel plant genome DNA extraction kit produced by Tiangen biochemical technology (Beijing) limited company. The specific operation is carried out according to the instruction of the novel plant genome DNA extraction kit.
42 samples to be tested are amplified by using the labeled primer provided in the first embodiment of the invention. Specifically, the amplification system comprises: 50ng of DNA fragment of the sample to be tested, 0.5. Mu.L of 10mM dNTP (deoxynucleotide triphosphate), 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 2.5. Mu.L of Tap Buffer, 0.2. Mu.L of Taq enzyme, and ddH was added 2 O made up the amplification system to 20. Mu.L, the amplification procedure included: 3min at 95 ℃; (95 ℃ C. 30sec,60 ℃ C. 30 sec). Times.30 cycles; and at 72℃for 6min. And (5) carrying out second-generation high-throughput sequencing on the amplified product to obtain sequencing data. Sequencing data were aligned to the rubber tree reference genome (NCBI: PRJNA 587314) using Bowtie2 software to obtain sequencing results for each sample tested.
Sequencing results were analyzed using Microsoft Office Excel software for a total of 5 different sequence types. The 5 different sequence types were designated a, b, c, d and e, respectively, as shown in table 2.
Table 2 shows the sequences obtained by amplifying and sequencing 42 samples to be tested by using the molecular marker primer pair
The software DNASTAR lasergene is used for comparing 5 different sequence types, the comparison result is shown in fig. 1, 8 base sites of 5 different sequences have differences, and the specific sequence differences include: 7 th base position G/A, 11 th base position G/T, 33 th base position A/G, 44 th base position C/T, 45 th base position G/A, 124 th base position A/T, 137 th base position A/C, 158 th base position G/T/C.
Among 42 samples to be tested provided in this embodiment, 5 samples to be tested can be effectively distinguished by using the primer pair provided in this embodiment, and different sequence combinations of the 5 samples to be tested are shown in table 3.
Table 3 shows different sequence combinations of 5 samples to be tested
| Sequence number | Sample name to be measured | Sequence combination |
| 1 | IAN873 | ae |
| 2 | GT1 | bc |
| 3 | Cloud computing 774 | bcd |
| 4 | Cloud grinding 3042 | bce |
| 5 | Thermal reclamation 516 | de |
As can be seen from table 3, the 5 samples to be tested have different sequence combinations, and it can be seen that the primer provided in the first embodiment of the present invention can amplify the sequence of the cloud cover 3042, and the sequence combination is bce, and the distinction between the rubber tree IAN873, GT1, cloud cover 774 and thermal reclamation 516 can be realized according to the different sequence combinations.
Accuracy analysis of identification of rubber tree molecular marker primers
In the embodiment, three independent experiments are performed by adopting different personnel, different batches of reagents and different laboratories, so that the identification of different batches of rubber trees is simulated, and the high reproducibility means that the identification results of different laboratories can be accurately compared with each other.
Reproducibility experiments are carried out on 5 rubber tree samples to be tested, and the 5 samples to be tested are specifically as follows: three repeated experiments are respectively carried out on each sample to be tested by cloud grinding 3042, IAN873, GT1, cloud grinding 774 and hot reclamation 516, and the sequence combination of each result is recorded, and the specific results are shown in Table 4.
Table 4 shows the results of three replicates
As can be seen from Table 4, the sequence combinations of the 3 replicates were identical. Therefore, the accuracy of the molecular marker primer identification provided by the embodiment of the invention is 100%. Therefore, the embodiment of the invention has higher consistency in the variety identification conclusion among different laboratories or different batches of the same laboratory, so that parallel experiments are not needed to reduce experimental errors, and great convenience is provided for the variety identification of the rubber tree.
The embodiment of the invention provides a molecular marker primer, a kit and application for identifying the rubber tree cloud research 3042, and the molecular marker primer and the kit can be used for distinguishing varieties by using a sequencing result of markers, such as the difference, substitution, insertion, deletion, repetition and the like of one or more bases among different sequences, so that the identification of the cloud research 3042 is realized, the rubber tree cloud research 3042 is an excellent triploid, meanwhile, the rubber tree IAN873, the GT1, the cloud research 774 and the thermal reclamation 516 can be distinguished, the identification result is not influenced by environment and other human factors, and the identification of the rubber tree varieties is further accurately carried out.
The foregoing description of the preferred embodiments of the present disclosure is provided for the purpose of illustration only, and is not intended to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, alternatives, and alternatives falling within the spirit and principles of the disclosure.
Claims (4)
1. The molecular marker primer identified by the rubber tree cloud-ground 3042 is characterized by comprising an upstream primer and a downstream primer, wherein the upstream primer is shown as SEQ ID NO:1, wherein the downstream primer is shown as SEQ ID NO: 2.
2. A kit for identifying rubbertree cloud-research 3042, comprising the molecular marker primer of claim 1.
3. An application of a molecular marker primer identified by rubber tree cloud-research 3042, which is characterized by comprising the following steps: the molecular marker primer is used for identifying the rubber tree cloud research 3042, and comprises an upstream primer and a downstream primer, wherein the upstream primer is shown as SEQ ID NO in a sequence table: 1, wherein the downstream primer is shown as SEQ ID NO: 2.
4. The application of claim 3, wherein the application further comprises: the molecular marker primer can also pair: rubber trees IAN873, GT1, cloud-ground 774, and hot reclamation 516 are distinguished.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310663966.2A CN116622892A (en) | 2023-06-01 | 2023-06-01 | Molecular marker primer and kit for identifying rubber tree cloud-ground 3042 and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310663966.2A CN116622892A (en) | 2023-06-01 | 2023-06-01 | Molecular marker primer and kit for identifying rubber tree cloud-ground 3042 and application |
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| Publication Number | Publication Date |
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| CN116622892A true CN116622892A (en) | 2023-08-22 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202310663966.2A Pending CN116622892A (en) | 2023-06-01 | 2023-06-01 | Molecular marker primer and kit for identifying rubber tree cloud-ground 3042 and application |
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| CN (1) | CN116622892A (en) |
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- 2023-06-01 CN CN202310663966.2A patent/CN116622892A/en active Pending
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