Astragalus sinicus EST-SSR (expressed sequence tag-simple sequence repeat) primer group and application
By designing the EST-SSR primer set of Estyrofoam, combining fluorescent labeling and multiple rounds of screening, the problem of Estyrofoam variety identification was solved, efficient and stable variety distinction and genetic diversity analysis were achieved, and the technical gap in molecular marking of green manure crops was filled.
Patent Information
- Application Number
- CN202510437112.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2025-07-08
AI Technical Summary
It is difficult to distinguish the identification of the species of cymbidium, especially in cross-pollinated plants, individual genotypes are hybridized within the varieties. The morphological identification is greatly affected by the environment and has poor molecular marker stability.
The EST-SSR primer set of cymbidium was designed, and the primer pairs with high specificity and polymorphism were screened for genetic diversity analysis and variety identification through fluorescent labeling and PCR amplification, combined with agarose gel initial screening and polyacrylamide gel re-sieve.
It has achieved efficient and stable genetic differences in different species of cymbidium species, solved the problem of heterozygous in varieties caused by cross-pollination, and provided an efficient and reliable germplasm resource identification tool.
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Figure CN120272632A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of molecular identification, and particularly relates to an EST-SSR primer set of Astragalus sinicus and its application. Background Art
[0002] Astragalus sinicus L. is an annual herbaceous plant of the genus Astragalus in the legume family and is the most important winter green manure in paddy fields in southern China. Since Astragalus sinicus is a cross-pollinated plant, the individual genotypes within the variety are heterozygous, and it is difficult to identify the variety. It is very difficult to distinguish different varieties of Astragalus sinicus from morphology, and the results are easily affected by environmental factors; molecular markers reveal genetic variations at the DNA level, are less affected by external factors, and the results are stable and reliable.
[0003] At present, simple sequence repeat (SSR) is a molecular marker method widely used for variety identification, which is determined by different nucleotide repeat numbers. According to the source of SSR, it can be divided into two types: genomic SSR and EST-SSR (expressed sequence tag, EST). Compared with genomic SSR, ESR-SSR exists in the exons of genes and is more conservative and stable. EST-SSR markers are developed based on transcriptome sequencing technology and have the characteristics of good repeatability, high polymorphism, strong stability, and large information content. Summary of the Invention
[0004] The purpose of the present invention is to provide an EST-SSR primer set of Astragalus sinicus and its application, providing an effective means for genetic identification of Astragalus sinicus.
[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:
[0006] The present invention provides an EST-SSR primer set of Astragalus sinicus, and the primer set includes one or more of primer pair P3, primer pair P4, primer pair P5, and primer pair P6;
[0007] The primer pair P3 contains an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6;
[0008] The primer pair P4 contains an upstream primer shown in SEQ ID NO.7 and a downstream primer shown in SEQ ID NO.8;
[0009] The primer pair P5 contains an upstream primer shown in SEQ ID NO.9 and a downstream primer shown in SEQ ID NO.10;
[0010] The primer pair P6 contains an upstream primer as shown in SEQ ID NO.11 and a downstream primer as shown in SEQ ID NO.12.
[0011] The present invention also provides a kit for Astragalus sinicus EST-SSR markers, and the kit includes the above primer set provided by the present invention;
[0012] Preferably, the primer set is labeled with a fluorescent group.
[0013] Preferably, the kit further includes a PCR amplification system.
[0014] Preferably, the PCR amplification system includes: 2 μL of DNA solution, 1 μL of 10 nmol / L upstream primer, 1 μL of 10 nmol / L downstream primer, 6 μL of pure water, and 10 μL of 2×PCR Mix.
[0015] The present invention also provides the application of the above primer set or kit in the genetic diversity analysis of Astragalus sinicus germplasm resources.
[0016] The present invention also provides the application of the above primer set or kit in the analysis of the genetic relationship of Astragalus sinicus.
[0017] The present invention also provides the application of the above primer set or kit in the identification of Astragalus sinicus varieties.
[0018] Preferably, the usage method of the kit includes the following steps:
[0019] Take the Astragalus sinicus sample to be identified and extract genomic DNA;
[0020] Using the genomic DNA as a template, perform PCR amplification with the above primer set;
[0021] According to the PCR amplification products, perform genetic diversity analysis of Astragalus sinicus germplasm resources, analysis of the genetic relationship of Astragalus sinicus, or identification of Astragalus sinicus varieties.
[0022] Preferably, the genomic DNA is extracted by the CTAB method;
[0023] In the CTAB method, the CTAB extraction solution contains 0.5-2% β-mercaptoethanol and 1-3% PVP K30.
[0024] Preferably, the reaction program of the PCR amplification includes: pre-denaturation at 95°C for 7 min; then 30 cycles, each cycle with denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; finally, extension at 72°C for 7 min.
[0025] The beneficial effects of the present invention:
[0026] The EST-SSR primers provided by the present invention are precisely designed based on the transcriptome data of Astragalus sinicus, and combined with a two-step screening process (primary screening by agarose gel and secondary screening by polyacrylamide gel), achieving high specificity, polymorphism, and amplification stability. They can effectively distinguish the genetic differences of different Astragalus sinicus varieties and can be used to solve the problem of intra-varietal heterozygosity caused by cross-pollination.
[0027] The technical system provided by the present invention combines economy and practicality. Through low-cost and high-throughput fluorescent SSR markers, it provides an efficient and reliable tool for the identification of Astragalus sinicus germplasm resources, genetic diversity analysis, and molecular breeding, filling the technical gap in the development of molecular markers for green manure crops and having important application value for the genetic improvement and resource management of Astragalus sinicus. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 It is the 1.2% agarose gel electrophoresis diagram of the primer amplification product;
[0029] Figure 2 It is the comparison result of two staining methods for polyacrylamide gel. Left figure: silver nitrate staining method for polyacrylamide gel; right figure: fluorescent nucleic acid dye staining method for polyacrylamide gel.
[0030] Figure 3 It is to screen the primers again by polyacrylamide gel method. Figure A: The amplified product band of primer P5 is clear and the size of the target band is correct; Figure B: The amplified product band of primer P9 is not clear.
[0031] Figure 4 It is the amplification map of primer P3 in two Astragalus sinicus varieties;
[0032] Figure 5 It is the amplification map of primer P4 in two Astragalus sinicus varieties;
[0033] Figure 6 It is the amplification map of primer P5 in two Astragalus sinicus varieties;
[0034] Figure 7 It is the amplification map of primer P6 in two Astragalus sinicus varieties. DETAILED DESCRIPTION OF THE INVENTION
[0035] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0036] Embodiment
[0037] Genomic DNA extraction and detection: The CTAB method was used to extract Astragalus sinicus DNA. Since Astragalus sinicus plants contain relatively more polyphenolic substances, 1% β-mercaptoethanol and 2% PVPK30, antioxidants, were added to the CTAB extraction solution when extracting Astragalus sinicus DNA. For each material, 50 plump and shiny seeds were selected and rubbed with 400-grit sandpaper, and then the seed germination boxes were germinated in the greenhouse. After 7 days of growth, 30 single-plant samples of each Astragalus sinicus variety were taken for DNA extraction. The DNA concentration was measured using Nanodrop2000, and samples with OD 260 / OD 280 less than 1.7 or greater than 2.0 were re-purified. The DNA concentration was adjusted to 50 nmol / L for subsequent experiments.
[0038] SSR locus screening and primer design: The transcriptome of Astragalus sinicus was derived from the raw transcriptome data (PRJNA728570) published on NCBI (http: / / www.ncbi.nlm.nih.gov). The MISA software was used to search for SSR loci in the transcriptome. The search criteria were that mononucleotide repeats were more than 10 times, dinucleotide repeats were more than 6 times, and trinucleotide, tetranucleotide, and pentanucleotide repeats were more than 5 times. SSR loci meeting the above requirements were screened out, and then primers were designed based on the information of each locus. Ten pairs of SSR primers were obtained and sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis, as shown in Table 1 below.
[0039] Table 1 The 10 pairs of EST-SSR primers obtained
[0040]
[0041] Preliminary screening of SSR primers: The above 10 pairs of primers were preliminarily verified. The PCR reaction program was: pre-denaturation at 95°C for 7 min; then 30 cycles, with each cycle consisting of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; finally, extension at 72°C for 7 min. The PCR amplification products were detected using 1.2% agarose gel electrophoresis, and SSR primers with amplified bands and band sizes consistent with the predicted values were selected.
[0042] Re-screening of SSR primers: Prepare a 6% polyacrylamide gel and perform polyacrylamide gel electrophoresis on the products amplified by the preliminarily screened primers. (1) Mix samples. Take 2 μL of the PCR product system and mix it with 2×TBE urea loading buffer. Treat it at 95 °C for 5 min on a PCR instrument, pre-cool it on ice, and then perform electrophoresis. (2) Pre-electrophoresis. Fix the glass plate (with the notched glass plate facing inward) on the vertical electrophoresis tank, and then pour 1×TBE buffer into the vertical electrophoresis tank, ensuring that the 1×TBE buffer covers the top of the electrophoresis tank and all the bubbles at the bottom of the glass plate are expelled (if there are bubbles, carefully shake to remove the bubbles). Pre-electrophorese at 120 V for 30 min. (3) Electrophoresis. Set the voltage to 60 V for 15 min to concentrate the bands. Subsequently, set the voltage to 120 V for 1 h. (4) Staining. Dilute 15 μL of Gelred 10,000× stock solution into 0.1 M NaCl to prepare a 3× staining solution. Carefully place the gel into a suitable container and slowly add enough 3× staining solution to cover the gel. Shake and stain at room temperature for about 10 min. (5) Re-screening of primers. Screen the primers with clear and accurate target bands.
[0043] After two screenings of capillary gel electrophoresis SSR, the screening primers are labeled with FAM fluorescent molecules (synthesized by Sangon Biotech (Shanghai) Co., Ltd.). Use these primers to amplify the DNA of all milk vetch varieties, and the amplified products are detected and typed using the capillary automatic fluorescence electrophoresis system ABI3730XL. Use GeneMarker V1.91 software to interpret the PCR amplification results and count the sizes of the obtained electrophoresis fragments.
[0044] The experimental results are as follows:
[0045] Perform annealing temperature gradient PCR tests on 10 pairs of primers, and set the annealing temperature range from 55 to 65 °C. The results ( Figure 1 ) show that primers P1 and P10 have no amplification products, and the products amplified by primer P8 do not match the expected target product size. Therefore, these three primers are discarded.
[0046] Nucleic acid fluorescence dye staining method for polyacrylamide gel: The commonly used staining method for polyacrylamide gel is silver staining. In this method, first pour the prepared 10% glacial acetic acid into the tray to cover the gel and shake it evenly on a shaker for 20 min for fixation. After rinsing the gel block twice with ultrapure water, then pour in the staining solution (500 ml of ultrapure water plus 3 ml of formaldehyde and 0.5 g of AgNO3), shake and stain on a shaker for 15 min, then rinse twice with ultrapure water, add 1.5% NaOH developing solution (500 ml of developing solution plus 3 ml of formaldehyde), after the bands are clear, pour out the developing solution, rinse twice with ultrapure water, and take pictures for recording. This method takes nearly 1 h, while when using nucleic acid fluorescent fuel for color development, pictures can be taken in about 15 min. This method greatly saves the operation time. See the comparison result diagram inFigure 2 。
[0047] Re-screening of EST-SSR primers
[0048] Next, use the 7 pairs of screened primers to amplify different Astragalus sinicus varieties, and then separate the amplification products by polyacrylamide gel electrophoresis. Keep the primers with clear amplification bands, such as P5( Figure 3 A); discard those with unclear bands, such as P9( Figure 3 B). After two screenings, 4 pairs of primers were finally left out of 10 pairs. Finally, 4 pairs of primers were screened out, namely P3, P4, P5, and P6. These four pairs amplified different bands, showing polymorphism, and at the same time the bands were clear.
[0049] Fluorescent labeling of EST-SS primers and capillary gel electrophoresis: Modify these 4 pairs of primers with the fluorescent molecule FAM, then perform PCR amplification on Astragalus sinicus varieties, and detect the amplification products by capillary fluorescence electrophoresis gel to accurately read the sizes of the amplification bands of different primers in different Astragalus sinicus varieties. The obtained data can be used for subsequent analysis. Analyze the amplification polymorphism of 4 pairs of primers using two Astragalus sinicus varieties, Minzi No. 7 and Xinzi No. 1. From Figure 4 it can be seen that the sizes of the amplification products of primer P3 for Minzi No. 7 are 188 and 191 respectively; the sizes of the amplification products for Xinzi No. 1 are 188 respectively; there are differences in the sizes of the amplification products between the two varieties, which can distinguish the two varieties of Minzi No. 7 and Xinzi No. 1. Similarly, the sizes of the amplification products of primer P4 for Minzi No. 7 are 105 and 108 respectively, and the sizes of the amplification products for Xinzi No. 1 are 108 and 111 respectively( Figure 5 ); the sizes of the amplification products of primer P5 for Minzi No. 7 are 212 and 215 respectively, and the sizes of the amplification products for Xinzi No. 1 are 215 and 232 respectively( Figure 6 ); the sizes of the amplification products of primer P6 for Minzi No. 7 are 175 and 178 respectively, and the sizes of the amplification products for Xinzi No. 1 are 178 and 187 respectively( Figure 7 ). In summary, using capillary gel electrophoresis, the sizes of the amplification products of 4 pairs of primers can be accurately read, and the amplification products of the 4 pairs of primers all show polymorphism, which is suitable for distinguishing Astragalus sinicus varieties and constructing the fingerprint map and core germplasm bank of Astragalus sinicus.
[0050] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. An EST-SSR primer set for Astragalus sinicus, characterized in that, The primer set includes one or more of primer pair P3, primer pair P4, primer pair P5, and primer pair P6; The primer pair P3 contains an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6; The primer pair P4 contains an upstream primer shown in SEQ ID NO.7 and a downstream primer shown in SEQ ID NO.8; The primer pair P5 contains an upstream primer shown in SEQ ID NO.9 and a downstream primer shown in SEQ ID NO.10; The primer pair P6 contains an upstream primer shown in SEQ ID NO.11 and a downstream primer shown in SEQ ID NO.
12.
2. A kit for the EST-SSR markers of Astragalus sinicus, characterized in that, The kit includes the primer set described in claim 1; Preferably, the primer set is labeled with a fluorescent group.
3. The kit according to claim 2, wherein The kit further includes a PCR amplification system.
4. The kit according to claim 3, wherein The PCR amplification system includes: 2 μL of DNA solution, 1 μL of 10 nmol / L upstream primer, 1 μL of 10 nmol / L downstream primer, 6 μL of pure water, and 10 μL of 2×PCR Mix.
5. Use of the primer set described in claim 1 or the kit described in any one of claims 2 to 4 in the analysis of genetic diversity of milk vetch germplasm resources.
6. Use of the primer set described in claim 1 or the kit described in any one of claims 2 to 4 in the analysis of genetic relationships of milk vetch.
7. Use of the primer set described in claim 1 or the kit described in any one of claims 2 to 4 in the identification of milk vetch varieties.
8. The application according to any one of claims 5 to 7, characterized in that, The usage method of the kit includes the following steps: Take the milk vetch sample to be identified and extract genomic DNA; Using the genomic DNA as a template, perform PCR amplification with the primer set described in claim 1; Based on the PCR amplification product, perform analysis of genetic diversity of milk vetch germplasm resources, analysis of genetic relationships of milk vetch, or identification of milk vetch varieties.
9. The application according to claim 8, wherein The genomic DNA is extracted by the CTAB method; In the CTAB method, the CTAB extraction solution contains 0.5 - 2% β-mercaptoethanol and 1 - 3% PVP K30.
10. The application according to claim 8, characterized in that, The reaction program of the PCR amplification includes: pre-denaturation at 95°C for 7 min; then 30 cycles, each cycle with denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; finally, extension at 72°C for 7 min.