Primer group and kit for identifying astragalus mongholicus and method for identifying astragalus mongholicus

Abstract

The invention provides a primer group for identifying astragalus mongholicus and an identification method of astragalus mongholicus, and belongs to the technical field of molecular markers, the primer group comprises a primer 186781F and a primer 186781R; the nucleotide sequence of the 186781F is as shown in SEQ ID NO. 1, and the nucleotide sequence of the 186781R is as shown in SEQ ID NO. 2. The primer group provided by the invention is designed aiming at the SSR molecular marker, and the SSR molecular marker has the advantages of uniform distribution, rich polymorphism, good amplification repeatability, clear banding pattern and the like in an astragalus membranaceus genome, and is suitable for detection of a common denaturing polyacrylamide gel electrophoresis detection platform and a capillary fluorescence detection platform; the method can be further applied to the research fields of astragalus genetic diversity analysis, variety identification, DNA fingerprint construction, molecular marker-assisted breeding and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and in particular relates to a primer set and a kit for identifying Mongolian astragalus and a method for identifying Mongolian astragalus. Background Art

[0002] Astragalus, originally called "Huangqi", is sweet in taste and slightly warm in nature. It is listed as a top-grade medicine in Shennong's Herbal Classic and is a commonly used tonic medicine in clinical practice. Astragalus has the effects of replenishing qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, promoting fluid and nourishing blood, relieving stagnation and relieving arthritis, expelling toxins and draining pus, and healing sores and promoting tissue regeneration. Modern medical research shows that Astragalus can enhance the body's immunity, resist aging, resist viruses, and strengthen the heart.

[0003] There are two kinds of genuine Astragalus: Mongolian Astragalus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao and Astragalus membranaceus (Fisch.) Bge., both of which are diploid plants. Traditional Chinese medicine identification methods, such as trait identification and microscopic identification, have inherent defects such as low accuracy, large subjective influence, and low versatility. They can no longer meet the needs of the booming Chinese medicine modernization market. How to use a simple, accurate and rapid method to distinguish the authenticity and distinguish the quality has become a difficult problem for many Chinese medicine researchers.

[0004] Molecular markers are the fourth generation of genetic markers after morphological markers, cytological markers, and protein markers. They have many advantages, such as a large number of markers, markers not being affected by the environment, simple operation, and low development costs.

[0005] Molecular markers such as SSR, AFLP, and RAPD can all be used to analyze the genetic diversity of Astragalus. Among them, SSR molecular markers have the following advantages over other markers: (1) they are abundant and widely distributed throughout the genome; (2) they have good experimental repeatability and reliable results; (3) they have more allelic variation; (4) they have low requirements for DNA quality, require less DNA, and do not require the use of isotopes; (5) they are co-dominant markers and can identify the genotypes of heterozygotes and homozygotes. Therefore, the development of SSR molecular markers can present as many genetic differences as possible in these samples at the molecular level, thereby eliminating the false and retaining the true, and eliminating the inferior and retaining the good. In addition, the development of genetic maps, gene positioning, genetic diversity analysis, and improved variety breeding based on molecular markers will help further promote the development of Astragalus towards high quality, standardization, and industrialization.

[0006] Prior art Chinese patent CN109735643A (published on May 10, 2019) discloses a method for identifying or assisting in identifying Astragalus based on single nucleotide polymorphism markers, which includes the following steps: 1) extracting DNA of the Astragalus to be tested as a template; 2) through sequencing, if the 476th position of the ITS sequence is a T base, then the Astragalus to be tested is Astragalus membranaceus, and if the 476th position of the ITS sequence is a C base, then the Astragalus to be tested is Astragalus mongolicus. This technology mainly uses single nucleotide mutations in the ITS sequence to identify Astragalus, which requires sequencing. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a primer set, a kit and an identification method of Mongolian Astragalus; the primer set provided by the present invention is designed for SSR molecular markers, and SSR molecular markers have the advantages of uniform distribution, rich polymorphism, good amplification repeatability, clear banding, etc. in the Astragalus genome, and are suitable for detection by ordinary denaturing polyacrylamide gel electrophoresis detection platform and capillary fluorescence detection platform, and can be further applied to research fields such as genetic diversity analysis of Astragalus, variety identification, DNA fingerprint map construction, molecular marker-assisted breeding, etc.

[0008] The invention provides a primer set for identifying Mongolian Astragalus, comprising primer 18678_1F and primer 18678_1R; the nucleotide sequence of the 18678_1F is shown in SEQ ID NO.1, and the nucleotide sequence of the 18678_1R is shown in SEQ ID NO.2.

[0009] The invention provides a kit for identifying Mongolian Astragalus, comprising the primer set and a detection reagent.

[0010] Preferably, the detection reagent is a PCR amplification reagent.

[0011] The present invention provides a method for identifying Astragalus mongolica using the primer set, comprising the following steps:

[0012] 1) Extracting genomic DNA from the sample to be tested;

[0013] 2) using the genomic DNA obtained in step 1) as a template, and performing PCR amplification with the primer set to obtain an amplified product;

[0014] 3) Detecting the amplified product obtained in step 2). If the amplified product has only a single 144 bp sequence, the sample to be tested is Astragalus mongolica.

[0015] Preferably, in step 1), the material for extracting genomic DNA of the sample to be tested is seedling leaves.

[0016] Preferably, the concentration of the template in step 2) is 10-20 ng / μl.

[0017] Preferably, the concentration of the primer in step 2) is 8-12 μM.

[0018] Preferably, the PCR amplification system in step 2) is calculated as 20 μl and includes the following components: 1 μl of template, 0.5 μl of primer 18678_1F, 0.5 μl of primer 18678_1R, 10 μl of 2×MIX, and 8 μl of ddH2O;

[0019] Step 2) pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s for 15 cycles; pre-denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s for 20 cycles; and finally extension at 72°C for 7 min.

[0020] Preferably, the method for detecting the amplification product includes polyacrylamide gel electrophoresis or fluorescent labeling capillary electrophoresis.

[0021] The present invention also provides application of the primer set or the kit in astragalus assisted breeding.

[0022] Compared with the prior art, the present invention has the following beneficial effects: the primer set for identifying Mongolian Astragalus provided by the present invention is an SSR primer set, and the present invention extracts DNA from Astragalus germplasm resources, constructs a genomic library by mixed sampling, sequences, and searches for SSR sites; primers are designed for the searched SSR sequences, and primer polymorphism is detected to obtain the primer set provided by the present invention; the primer set has good amplification repeatability and clear banding; and can be further applied to research fields such as genetic diversity analysis of Astragalus, variety identification, DNA fingerprint map construction, and molecular marker-assisted breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Electrophoresis of the amplified product obtained by PCR amplification using primers Figure 1 (104923~19262);

[0024] Figure 2 Electrophoresis of the amplified product obtained by PCR amplification using primers Figure 2 (20195~26222);

[0025] Figure 3 Electrophoresis of the amplified product obtained by PCR amplification using primers Figure 3 (26818~47749)

[0026] Figure 4 Electrophoresis of the amplified product obtained by PCR amplification using primers Figure 4 (483~900)

[0027] Figure 5 Electrophoresis of the amplified product obtained by PCR amplification using primers Figure 5 (9129~1859). DETAILED DESCRIPTION

[0028] The present invention provides a primer set for identifying Mongolian Astragalus, comprising primer 18678_1F and primer 18678_1R; the nucleotide sequence of the 18678_1F is shown in SEQ ID NO.1, and the nucleotide sequence of the 18678_1R is shown in SEQ ID NO.2; specifically as shown in Table 1.

[0029] Table 1 Sequences of primer sets

[0030]

[0031]

[0032] The invention also provides a kit for identifying Mongolian Astragalus, comprising the primer set and a detection reagent.

[0033] In the present invention, the detection reagent is preferably a PCR amplification reagent. The present invention has no special limitation on the source of the PCR amplification reagent, and conventional commercial products in the art can be used.

[0034] The present invention provides a method for identifying Astragalus mongolica using the primer set, comprising the following steps:

[0035] 1) Extracting genomic DNA from the sample to be tested;

[0036] 2) using the genomic DNA obtained in step 1) as a template, and performing PCR amplification with the primer set to obtain an amplified product;

[0037] 3) Detecting the amplified product obtained in step 2). If the amplified product has only a single 144 bp sequence, the sample to be tested is Astragalus mongolica.

[0038] In the present invention, the genomic DNA of the sample to be tested is first extracted. The material for extracting the genomic DNA of the sample to be tested is preferably seedling leaves. The present invention has no special limitation on the method for extracting the genomic DNA, and the plant genomic DNA extraction method known in the art can be used.

[0039] After obtaining the genomic DNA, the present invention uses the obtained genomic DNA as a template and performs PCR amplification with the primer set to obtain an amplified product. In the present invention, the concentration of the template is preferably 10 to 20 ng / μl, more preferably 12 to 18 ng / μl, and most preferably 15 ng / μl, and the concentration of the primer is preferably 8 to 12 μM, more preferably 9 to 11 μM, and even more preferably 10 μM.

[0040] In the present invention, the PCR amplification system is calculated as 20 μl and includes the following components: 1 μl of template, 0.5 μl of primer 18678_1F, 0.5 μl of primer 18678_1R, 10 μl of 2×MIX, and 8 μl of ddH2O. The PCR amplification program is preferably 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, for 15 cycles; 95°C pre-denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 30 s, for 20 cycles; and finally 72°C extension for 7 min.

[0041] After obtaining the amplification product, the present invention detects the amplification product. If the amplification product has only a single 144bp sequence, the sample to be tested is Astragalus mongolica. In the present invention, the method for detecting the amplification product includes polyacrylamide gel electrophoresis or fluorescent labeling capillary electrophoresis.

[0042] The present invention also provides application of the primer set or the kit in astragalus assisted breeding.

[0043] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] Example 1

[0045] Obtaining genome sequences: DNA was extracted from the nine representative Astragalus germplasm resources selected (as shown in Table 2), and the genomic library was constructed by mixing samples. The core of the SSR and the conserved sequence regions at both ends were enriched by magnetic beads for sequencing; this part of the work was entrusted to Nanjing Paisonno Technology Co., Ltd., and the project name was SSR molecular marker development (YFnj20224223).

[0046] Table 2 Representative germplasm resources of Astragalus

[0047] Serial number name Origin A1 1 Mongolian Astragalus green with hair Guaner Township, Hunyuan A2 2 Mongolian Astragalus Green Hairless Hunyuan Peicun Township A3 3 Mongolian Astragalus green hairless flower tip red Hunyuan Peicun Township A4 4 Mongolian Astragalus Red Hairless Hunyuan Shagetuo Town A5 5 Mongolian Astragalus Red Hairy Hunyuan Celadon Kiln Town A6 6 Astragalus membranaceus red hairy Wuzhai Heyeping A7 7 Astragalus membranaceus green hairy Hezheng County, Gansu A8 8 Astragalus membranaceus green hairless Hezheng County, Gansu A9 9 Astragalus membranaceus red hairless Hezheng County, Gansu

[0048] (2) Search for genomic sequences containing SSR: Search for SSR sequences in the sequenced sequences and count the search results of SSR.

[0049] (3) SSR primer design: SSRs were then clustered and their polymorphisms were evaluated. Primers were designed for polymorphic SSRs. The designed primers are shown in Table 3.

[0050] Table 3 List of SSR designed primers

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] SSR primer screening: DNA was extracted from the leaves of 5 Mongolian resources and 4 Astragalus membranaceus with large phenotypic differences from different sources (as shown in Table 2). The extracted DNA was amplified by PCR using the SSR primers designed in step (3). Then, polymorphism detection was performed using 6-8% polyacrylamide gel electrophoresis to screen effective SSR primers. The results are shown in Table 2. Figures 1 to 5 As shown, among the 100 pairs of primers listed, only the primer sets 18678_1F and 18678_1R provided by the present invention can clearly distinguish Astragalus mongolica from Astragalus membranaceus.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer set for identifying Astragalus mongolica, characterized in that: It includes primer 18678_1F and primer 18678_1R; the nucleotide sequence of the primer 18678_1F is shown in SEQ ID NO.1, and the nucleotide sequence of the primer 18678_1R is shown in SEQ ID NO.

2.

2. A kit for identifying Astragalus mongolica, characterized in that: It comprises the primer set and detection reagent described in claim 1.

3. The kit according to claim 2, characterized in that The detection reagent is a PCR amplification reagent.

4. A method for identifying Astragalus mongolica using the primer set of claim 1, characterized in that: The following steps are involved: 1) Extracting genomic DNA from the sample to be tested; 2) using the genomic DNA obtained in step 1) as a template, and performing PCR amplification using the primer set of claim 1 to obtain an amplified product; 3) Detecting the amplified product obtained in step 2). If the amplified product has only a single 144 bp sequence, the sample to be tested is Astragalus mongolica.

5. The method according to claim 4, characterized in that In step 1), the material for extracting genomic DNA of the sample to be tested is seedling leaves.

6. The method according to claim 4, characterized in that The concentration of the template in step 2) is 10-20 ng / μl.

7. The method according to claim 4, characterized in that Step 2) The concentration of the primer is 8-12 μM.

8. The method according to claim 6 or 7, characterized in that: Step 2) The PCR amplification system is 20 μl and includes the following components: template 1 μl, primer 18678_1F 0.5 μl, primer 18678_1R 0.5 μl, 2×MIX 10 μl, ddH2O 8 μl; Step 2) pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s for 15 cycles; pre-denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s for 20 cycles; and finally extension at 72°C for 7 min.

9. The method according to claim 4, characterized in that Methods for detecting the amplified products include polyacrylamide gel electrophoresis or fluorescent labeling capillary electrophoresis.

10. Use of the primer set according to claim 1 or the kit according to claim 2 in astragalus assisted breeding.

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