CN112553364A - ISSR-PCR molecular marker of Angelica crenulata and application thereof - Google Patents
ISSR-PCR molecular marker of Angelica crenulata and application thereof Download PDFInfo
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- 235000001287 Guettarda speciosa Nutrition 0.000 title claims description 23
- 239000003147 molecular marker Substances 0.000 title abstract description 11
- 244000061520 Angelica archangelica Species 0.000 title 1
- 241000125175 Angelica Species 0.000 claims description 22
- 238000000137 annealing Methods 0.000 claims description 9
- 238000004925 denaturation Methods 0.000 claims description 9
- 230000036425 denaturation Effects 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 238000012257 pre-denaturation Methods 0.000 claims description 9
- 238000003860 storage Methods 0.000 claims description 8
- 240000001810 Angelica gigas Species 0.000 claims description 6
- 235000018865 Angelica gigas Nutrition 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 241000196324 Embryophyta Species 0.000 claims description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 2
- 241000213006 Angelica dahurica Species 0.000 abstract 1
- 238000011282 treatment Methods 0.000 description 10
- 238000012408 PCR amplification Methods 0.000 description 9
- 239000003550 marker Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 241000382455 Angelica sinensis Species 0.000 description 7
- 238000011284 combination treatment Methods 0.000 description 7
- 238000005457 optimization Methods 0.000 description 7
- 238000013461 design Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
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- 238000004458 analytical method Methods 0.000 description 2
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- 239000000463 material Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000208173 Apiaceae Species 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
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- 238000003556 assay Methods 0.000 description 1
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- 238000012803 optimization experiment Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses an ISSR-PCR molecular marker for Chinese angelica with multiple teeth and application thereof, belonging to the technical field of molecular markers. By optimizing the reaction system of the UBC81, UBC807, UBC810, UBC816, UBC817 and UBC819 primers, the target band can be amplified quickly and accurately, and the reaction system is accurate and reliable, simple to operate, time and cost saving and clear and stable in amplified band.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to an ISSR-PCR molecular marker for angelica crenulata and application thereof.
Background
The angelica sinensis is a perennial herb medicinal plant of angelica of Umbelliferae, has the effects of dispelling wind and removing dampness, and relieving arthralgia and pain, and is one of the traditional Chinese medicinal materials in China. The angelica sinensis is artificially planted in Hubei, Chongqing, Gansu and Shaanxi. Currently, the current practice is. Research on the angelica crenulata mainly focuses on the active ingredients, clinical efficacy, cultivation technology and the like, and research on molecular markers of the angelica crenulata is lacked.
The molecular marking technology can reflect the genetic difference of species from DNA level, and is an effective method for researching genetic diversity. In recent years, the ISSR molecular marker technology has been widely used for variety identification of animals and plants due to its advantages such as simple operation, high polymorphism, and good reproducibility. However, the ISSR marking is a random marking method, and test parameters of different species and even the same species are different under different conditions, so that a scientific and reasonable ISSR-PCR reaction system is established, the optimal amount of each PCR component is searched, and theoretical basis can be provided for subsequent work of carrying out genetic diversity research, fine variety breeding and the like of the angelica sinensis.
Therefore, how to provide an ISSR-PCR molecular marker for Angelica gigas nakai and application thereof is a problem to be solved in the field.
Disclosure of Invention
The invention provides an ISSR-PCR molecular marker of angelica crenulata and application thereof, aiming at the problem that the existing research on the angelica crenulata is lack of molecular marker research.
In order to achieve the purpose, the invention adopts the following technical scheme:
an ISSR-PCR primer of angelica crenulata has the following sequence:
UBC812：GAGAGAGAGAGAGAGAA，SEQ ID NO.1；
UBC807：AGAGAGAGAGAGAGAGT，SEQ ID NO.2；
UBC810：GAGAGAGAGAGAGAGAT，SEQ ID NO.3；
UBC816：CACACACACACACACAT，SEQ ID NO.4；
UBC817：CACACACACACACACAA，SEQ ID NO.5；
UBC819：GTGTGTGTGTGTGTGTA，SEQ ID NO.6；
the reaction system is 20 mu L, which contains 2 XTaqMasterMix 9.8 mu L, template DNA 9.04ng, primer 0.325 mu M and the rest H2O；
Preferably, the used kit is a PCR kit 3.0 of Tianjing Sha series, a ready-to-use type, from Beijing Tianenzze Gene technology Co., Ltd;
the amplification procedure was: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 deg.C for 1 min; annealing at 53.9 ℃ for 40 s; stretching at 72 ℃ for 40s, and circulating for 40 times; extending at 72 deg.C for 5min, and storing at 4 deg.C;
an ISSR-PCR identification method for angelica crenulata is characterized by comprising the following steps:
1) collecting tender leaves of the angelica crenulata, and extracting plant group DNA;
2) detecting DNA by using 1% agarose gel electrophoresis;
3) and (2) amplifying the DNA sample extracted in the step 1) by using the ISSR-PCR primer through the ISSR-PCR reaction system.
In conclusion, the invention provides an ISSR-PCR molecular marker of angelica crenulata and application thereof. Compared with the prior art, the invention can obtain the following technical effects:
the invention establishes and optimizes the ISSR-PCR molecular marker reaction system of the Chinese angelica with 6 types of ISSR-PCR molecular markers of the Chinese angelica, and the method has the advantages of accuracy, reliability, simple operation, time and cost saving, clear and stable amplification strip and easy and rapid analysis and detection.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is the amplification results of an ISSR-PCR homogeneous design assay of the present invention; wherein M represents: marker; the primer is UBC 812; 1-12 are treatment combinations 1-12 of Table 1;
FIG. 2 shows ISSR-PCR amplification results of 2 × TaqMasterMix as a single factor; m represents: marker; the primer is UBC 812; 1-7 are treatment combinations 1-7 in table 2;
FIG. 3 shows the ISSR-PCR amplification result of the 2 XTAQQ Master Mix fine tuning of the present invention; m represents: marker; the primer is UBC 812; 1-11 are treatment combinations 1-11 in Table 3;
FIG. 4 shows ISSR-PCR amplification results with the primers of the present invention as a single factor; m represents: marker; the primer is UBC 812; 1-7 are treatment combinations 1-7 in Table 4;
FIG. 5 shows the ISSR-PCR amplification result of the primer trimming according to the present invention; m represents: marker; the primer is UBC 812; 1-9 are treatment combinations 1-9 in Table 5;
FIG. 6 shows ISSR-PCR amplification results of the DNA of the present invention as a single factor; m represents: marker; the primer is UBC 812; 1-7 are treatment combinations 1-7 in Table 6;
FIG. 7 shows the result of ISSR-PCR amplification of the present invention; m represents: marker; the primer is UBC 812; 1-9 are treatment combinations 1-9 in Table 5;
FIG. 8 shows ISSR-PCR amplification results of 7 different primers on Angelica gigas nakai samples; m represents: marker; the primers are UBC807, UBC810, UBC812, UBC814, UBC816, UBC817 and UBC 819; 1-7 is electrophoresis band of 7 primers to ISSR-PCR product of Angelica crenata sample.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Establishment and optimization of ISSR-PCR amplification system
1. Extraction of DNA
Cleaning a mortar with clear water, airing, adding absolute ethyl alcohol, igniting, cooling the mortar in the air, taking 500mg of collected angelica sinensis heavy teeth buds into the mortar, pouring a proper amount of liquid nitrogen, grinding for 3-5min when the liquid nitrogen submerges the sample at a height of 2-3cm, and extracting the angelica sinensis heavy teeth genome DNA by a kit method.
2. Establishment of ISSR-PCR amplification system
1) Uniform design test scheme of ISSR-PCR reaction system
The method adopts U12(43) to uniformly design a table, UBC840 as a primer and DNA of the angelica crenulata as a template, and utilizes 12 reaction systems in the table 2 to carry out optimized screening on ISSR-PCR, wherein the reaction system is 20 mu L, and the details are shown in the table 1.
The ISSR-PCR amplification program comprises the following steps: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 1min, annealing at 52.4 deg.C for 40s, extension at 72 deg.C for 40s, circulation for 40 times, re-extension at 72 deg.C for 5min, and storage at 4 deg.C.
TABLE 1
As a result, as shown in FIG. 1, amplified bands were observed in all treatments, including those obtained in treatments 5, 6, 7 and 9, which were less bright and less clear, and those obtained in treatments 3, 8 and 10, which were bright and clear and more numerous, which was preferable (FIG. 1). And comprehensively considering the effect and the cost, and selecting the condition No. 3 to carry out the following single-factor test.
Example 2
Optimization of 2 XTaq Master Mix dosage
According to the results of the uniform design experiment, UBC812 was used as a primer, genomic DNA of Angelica gigas was used as a template, and ISSR-PCR was performed in a 20. mu.L reaction system with the amounts of 2 XTAQASTERMix of 1. mu.L, 3. mu.L, 5. mu.L, 7. mu.L, 9. mu.L, 11. mu.L and 13. mu.L, respectively, and the primers, DNA and ddH2The amount of O used is shown in Table 2.
The ISSR-PCR amplification program comprises the following steps: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 1min, annealing at 52.4 deg.C for 40s, extension at 72 deg.C for 40s, circulation for 40 times, re-extension at 72 deg.C for 5min, and storage at 4 deg.C.
TABLE 2
The results of the single factor test showed that the amplification bands of Nos. 6 and 7 were numerous and bright (FIG. 2), and for a better treatment, the optimal condition was No. 6, and the 2 XTaq MasterMix dose quadratic optimization test was performed at the level of No. 6.
According to the results of the uniform design test, ISSR was performed using UBC812 as a primer and genomic DNA of Angelica gigas nakai as a template in amounts of 10. mu.l, 10.2. mu.l, 10.4. mu.l, 10.6. mu.l, 10.8. mu.l, 11. mu.l, 11.2. mu.l, 11.4. mu.l, 11.6. mu.l, 11.8. mu.l, and 12. mu.l, respectively, of 2 XTaq Master Mix in a 20. mu.l reaction systemPCR, primers, DNA and ddH2The amount of O used is shown in Table 3.
The ISSR-PCR amplification program comprises the following steps: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 1min, annealing at 52.4 deg.C for 40s, extension at 72 deg.C for 40s, circulation for 40 times, re-extension at 72 deg.C for 5min, and storage at 4 deg.C.
TABLE 3
The results showed that all the treated amplified bands were not smeared (FIG. 3), but the treated bands were different in brightness and contrast, and the condition No. 8 was found to be most preferable.
Example 3
Optimization of primer dosage
Using UBC812 as primer and genomic DNA of Angelica crenata as template, ISSR-PCR was performed in 20. mu.L reaction system with amounts of primers designed to be 1. mu.L, 2. mu.L, 3. mu.L, 7. mu.L, 5. mu.L, 6. mu.L, and 7. mu.L, 2 XTaq MasterMix, DNA, and ddH2The amounts of O used are shown in Table 4.
The ISSR-PCR amplification program comprises the following steps: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 1min, annealing at 52.4 deg.C for 40s, extension at 72 deg.C for 40s, circulation for 40 times, re-extension at 72 deg.C for 5min, and storage at 4 deg.C.
TABLE 4
The results showed that all treatments had amplified bands, but the bands No. 4, 5, 6, and 7 were numerous and bright (fig. 4), which was a relatively good treatment, and the primer usage secondary optimization test was performed based on the condition No. 4, which was relatively optimal for the condition No. 4.
Using UBC812 as a primer and genomic DNA of the primer as a template, primers were designed in a 20. mu.L reaction systemThe amounts of the substances were 3.2. mu.l, 3.4. mu.l, 3.6. mu.l, 3.8. mu.l, 4. mu.l, 4.2. mu.l, 4.4. mu.l, 4.6. mu.l, 4.8. mu.l, respectively, and ISSR-PCR, 2 XTAQASMasterMix, DNA and ddH were performed2The amounts of O used are shown in Table 5.
The ISSR-PCR amplification program comprises the following steps: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 1min, annealing at 52.4 deg.C for 40s, extension at 72 deg.C for 40s, circulation for 40 times, re-extension at 72 deg.C for 5min, and storage at 4 deg.C.
TABLE 5
All treatments were found to be banded, clear and stable (figure 5). The optimal amount of the primer for treatment 7 was determined by considering the amount and the resolution of the primer in combination.
Example 4
Optimization of DNA dosage
Using UBC812 as primer and genomic DNA of Angelica crenata as template, ISSR-PCR was performed in 20. mu.L reaction system with amounts of designed DNA of 0.5. mu.L, 1. mu.L, 1.5. mu.L, 2. mu.L, 2.5. mu.L, 3. mu.L, and 3.5. mu.L, 2 XTaq MasterMix, DNA, and ddH2The amounts of O used are shown in Table 6.
The ISSR-PCR amplification program comprises the following steps: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 1min, annealing at 52.4 deg.C for 40s, extension at 72 deg.C for 40s, circulation for 40 times, re-extension at 72 deg.C for 5min, and storage at 4 deg.C.
TABLE 6
The results show that all 7 treatments performed well (fig. 6), and after careful comparison the No. 6 condition was chosen for the DNA dose secondary optimization experiment.
Using UBC812 as primer and genomic DNA of Angelica crenata as template, ISSR-PCR was performed with amounts of designed DNA of 2.6. mu.l, 2.7. mu.l, 2.8. mu.l, 2.9. mu.l, 3. mu.l, 3.1. mu.l, 3.2. mu.l, 3.3. mu.l, and 3.4. mu.l, respectively, in a 20. mu.L reaction system, 2 XTAQASTAMASterMix, DNA, and ddH2The amounts of O used are shown in Table 7.
The ISSR-PCR amplification program comprises the following steps: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 1min, annealing at 52.4 deg.C for 40s, extension at 72 deg.C for 40s, circulation for 40 times, re-extension at 72 deg.C for 5min, and storage at 4 deg.C.
TABLE 7 DNA dosage Secondary optimization test treatment (μ L)
The ISSR-PCR amplification program comprises the following steps: in a 20. mu.L system, 2 XTaq Master Mix: 11.4 μ L, DNA: 9.04ng, primer: 0.325. mu. mol/L.
Example 5
Verification result of optimal dosage of each component of amplification system
7 different primers were selected: and carrying out ISSR-PCR verification on the optimized optimal reaction system by using DNA of the angelica crenulata as a template, wherein the DNA of the angelica crenulata is UBC807, UBC810, UBC812, UBC814, UBC816, UBC817 and UBC 819.
The results showed that the amplified band was clearly stabilized except for UBC814 (FIG. 8), indicating that the reaction system is suitable for ISSR analysis of Angelica gigas with primers UBC807, UBC810, UBC812, UBC816, UBC817, and UBC 819.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> institute of Chinese medicinal materials of academy of agricultural sciences of Hubei province
<120> Angelica crenata ISSR-PCR molecular marker and application thereof
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gagagagaga gagagaa 17
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agagagagag agagagt 17
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gagagagaga gagagat 17
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Claims (4)
1. An ISSR-PCR primer for angelica crenulata is characterized by comprising the following sequences:
UBC812：GAGAGAGAGAGAGAGAA，SEQ ID NO.1；
UBC807：AGAGAGAGAGAGAGAGT，SEQ ID NO.2；
UBC810：GAGAGAGAGAGAGAGAT，SEQ ID NO.3；
UBC816：CACACACACACACACAT，SEQ ID NO.4；
UBC817：CACACACACACACACAA，SEQ ID NO.5；
UBC819：GTGTGTGTGTGTGTGTA，SEQ ID NO.6。
2. a reaction system for the ISSR-PCR primer of claim 1, wherein the reaction system is 20 μ L, which contains 2 XTAQA MasterMix 9.8 μ L, template DNA 9.04ng, primer 0.325 μ M, and the rest is H2O。
3. The ISSR-PCR primer reaction system for Angelica gigas nakai of claim 2, wherein the amplification procedure is: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 deg.C for 1 min; annealing at 53.9 ℃ for 40 s; stretching at 72 ℃ for 40s, and circulating for 40 times; extension at 72 ℃ for 5min and final storage at 4 ℃.
4. An ISSR-PCR identification method for angelica crenulata is characterized by comprising the following steps:
1) collecting tender leaves of the angelica crenulata, and extracting plant group DNA;
2) detecting DNA by using 1% agarose gel electrophoresis;
3) amplifying the DNA sample extracted in step 1) by the ISSR-PCR reaction system of claims 2 and 3 using the ISSR-PCR primers of claim 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911256A (en) * | 2015-03-10 | 2015-09-16 | 泸州医学院 | Chinese angelica SCAR molecular marker, its identification method and specific primer pair |
| CN109055595A (en) * | 2018-09-07 | 2018-12-21 | 湖北省农业科学院中药材研究所 | A kind of Herba Epimedii ISSR-PCR molecule labelling method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911256A (en) * | 2015-03-10 | 2015-09-16 | 泸州医学院 | Chinese angelica SCAR molecular marker, its identification method and specific primer pair |
| CN109055595A (en) * | 2018-09-07 | 2018-12-21 | 湖北省农业科学院中药材研究所 | A kind of Herba Epimedii ISSR-PCR molecule labelling method |
Non-Patent Citations (4)
| Title |
|---|
| MAN LIU等: "Constructing a Core Collection of the Medicinal Plant Angelica biserrata Using Genetic and Metabolic Data", FRONT PLANT SCI, vol. 11, pages 10 - 12 * |
| 彭禄等: "基于ITS序列对独活17个种的分子鉴定", 中草药, no. 12, pages 135 - 140 * |
| 王巍等: "中药独活RAPD鉴别研究", 辽宁中医药大学学报, no. 03, pages 201 - 202 * |
| 黄晓慧等: "中国兰ISSR-PCR反应体系优化及引物筛选", 南方农业学报, vol. 49, no. 07, pages 1 * |
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Application publication date: 20210326 |