SNP (Single Nucleotide Polymorphism) molecular marker related to notoginsenoside R1 content in panax notoginseng and application
By developing the KASP primer combination for Panax notoginseng, the genotype detection of base 189765834 of chromosome 5 was used to solve the problem of long breeding cycle of Panax notoginseng, and early molecularly assisted selection of Panax notoginseng saponin R1 content was achieved, and breeding efficiency and accuracy were improved.
Patent Information
- Application Number
- CN202510906370.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-02
- Publication Date
- 2025-08-15
AI Technical Summary
The existing breeding cycle of Panax notoginseng is long and has low efficiency, and lacks early selection and identification methods, resulting in a breeding cycle of up to 15 to 20 years.
A SNP molecular marker based on KASP primer combination was developed, and the genotype detection was performed using the genotype detection of base 189765834 of chromosome Panax notoginseng genotype was selected to increase the content of panax notoginseng saponin R1, and genotyping was performed through KASP primer combination for PCR amplification and fluorescence signal reading.
Early molecular assisted selection of the R1 content trait of panaxanthin saponin was achieved, shortening the breeding cycle, improving breeding selection efficiency, reducing detection costs and improving the accuracy of detection results.
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Figure CN120485425A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of molecular genetic breeding, and particularly relates to a SNP molecular marker related to the content of notoginsenoside R1 in Panax notoginseng and its application. Background Art
[0002] Panax notoginseng (Burk.) FHChen is a perennial medicinal herb in the genus Ginseng and a traditional and precious Chinese medicinal material. Its main medicinal component is its underground parts (the taproot and cut ends). Modern pharmacological and clinical studies have shown that Panax notoginseng exhibits pharmacological activities in the immune, cardiovascular, and nervous systems, as well as in anti-tumor and anti-aging activities. Saponins are the primary active ingredients in Panax notoginseng, and their content and variety are important indicators for evaluating its quality.
[0003] Quality breeding is a core initiative in improving varieties. Research has shown that identifying superior allelic variants of key genes controlling important traits and developing allele-specific molecular markers can be effectively utilized in marker-assisted selection breeding. Current Panax notoginseng variety breeding still relies on traditional systematic selection. However, each generation requires at least three years, resulting in extremely slow phenotypic separation and population construction. Early selection and identification methods are also lacking, resulting in long breeding cycles and low efficiency. Cultivating a new variety can take 15 to 20 years.
[0004] In recent years, with the development of molecular biology, molecular genetic breeding has entered the genomic level. Molecular genetic breeding of medicinal plants based on molecular markers can greatly improve breeding efficiency and make it possible to target the molecular design breeding of effective ingredients in medicinal plants. As the third-generation DNA molecular marker, SNP has many advantages and is considered to be a molecular marker technology with great application prospects. KASP technology (Kompetitive Allele Specific PCR) is based on the specific matching of primer terminal bases for SNP typing and InDels detection. It can accurately judge the double alleles of SNPs and InDels at specific sites in a wide range of genomic DNA samples, and is highly stable and accurate.
[0005] Compared to traditional marker-assisted selection (MAS), high-throughput resequencing technology can generate large-scale SNP markers, facilitate accurate association analysis between markers and traits, and improve the efficiency of marker-assisted selection breeding. The content and type of saponins in Panax notoginseng are important indicators of its quality. Notoginseng saponin R1 is a characteristic component of the total saponins of Panax notoginseng, with benefits such as improving atherosclerosis, resisting thrombosis, lowering blood pressure, and improving myocardial ischemia. It also has a protective effect on the nervous system. Therefore, the content and type of saponins are the core objectives of variety breeding. Leveraging known saponin biosynthesis genes and abundant Panax notoginseng germplasm material, it is particularly important to identify SNPs significantly associated with notoginseng saponin R1 content in Panax notoginseng, develop KASP molecular markers for assisted breeding, and achieve early molecular-assisted selection for target traits to improve breeding efficiency. Summary of the Invention
[0006] The technical problem to be solved by the present invention is to provide a SNP molecular marker related to the content of notoginsenoside R1 in Panax notoginseng and its application.
[0007] The technical solution of the present invention is: a method for breeding Panax notoginseng with a high notoginseng saponin R1 content trait, detecting the genotype of the 189765834th base of chromosome 5 of the Panax notoginseng sample, and selecting Panax notoginseng samples with a CC genotype at this site as having a higher notoginseng saponin R1 content trait than other genotypes.
[0008] The invention discloses an application of a substance for detecting the genotype of a SNP site in assisted breeding for the notoginseng saponin R1 content trait. The SNP site is located at position 101 of the nucleotide sequence shown in SEQ ID No. 1 on chromosome 5 of Panax notoginseng. The site has a T / C polymorphism. The genotype of the site is detected, and Panax notoginseng with a CC genotype is selected, which has a higher notoginseng saponin R1 content trait than Panax notoginseng with a CT genotype or a TT genotype.
[0009] Furthermore, the substance for detecting the genotype of the SNP site is a KASP primer set, and the KASP primer set consists of primers shown in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4.
[0010] Furthermore, the method for detecting the genotype of the site is:
[0011] (1) Extracting genomic DNA from the Panax notoginseng sample to be tested;
[0012] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the KASP primer set described above.
[0013] (3) Read the fluorescence signal and complete genotyping.
[0014] Furthermore, the PCR amplification program is: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 20 seconds; annealing at 61-55°C for 1 minute, decreasing 0.6°C in each cycle, for 10 cycles; denaturation at 95°C for 20 seconds, annealing at 55°C for 1 minute, for 30 cycles.
[0015] Furthermore, the PCR amplification system is: 2 μL of 40-60 ng / μL DNA template; 5 μL of 2×KASP Mastermix; 0.5 μL of KASP primer set, 2.5 μL of DEPC water; in the KASP primer set, the volume ratio of the competitive primers shown in SEQ ID No. 2 and SEQ ID No. 3 and the universal reverse primer shown in SEQ ID No. 4 is 1:1:3.
[0016] A KASP primer set, wherein the KASP primer set consists of primers shown in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4.
[0017] A kit comprising the above-mentioned KASP primer set.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] The present invention provides a single nucleotide polymorphism (SNP) significantly associated with notoginsenoside R1 content in Panax notoginseng. This was determined using association analysis of 162 natural Panax notoginseng populations. Using the SNP locus as genotype data and notoginsenoside R1 content as phenotypic data, the genome-wide association survey (GWAS) analysis using the "BLINK" method was performed using BLINK C (Version 1.01) (parameter: --gwas). The SNP molecular marker SNP5-189765834, located at base 189765834 on chromosome Chr5, provides technical support for molecular marker-assisted breeding for notoginsenoside R1 content in Panax notoginseng.
[0020] The KASP primer combination developed in the present invention can directly and specifically distinguish and detect the T or C base at the mutation site of SNP 5-189765834. When using this KASP primer combination to identify the high and low levels of notoginseng saponin R1 content, it can clearly separate the two genotypes. The KASP primer combination developed in the present invention has excellent application value and can achieve pre-selection and molecular-assisted breeding for the notoginseng saponin R1 content trait in Panax notoginseng. It has important theoretical and practical guiding significance for the genetic improvement process of notoginseng saponin R1 content breeding and improving breeding selection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Manhattan and QQ-plot of GWAS results for ginsenoside R1 in Panax notoginseng;
[0022] Figure 2 This is a statistical diagram of the allelic variation and phenotypic significance of ginsenoside R1 content in Panax notoginseng; among them, **p<0.001, ****p<0.0001;
[0023] Figure 3 This is a statistical diagram of the allelic variation and phenotypic significance of notoginseng saponin R1 content in 92 Panax notoginseng samples; among them, **p<0.001, ****p<0.0001;. DETAILED DESCRIPTION
[0024] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.
[0025] Example 1 Obtaining the nucleotide mutation site (SNP) related to the content of notoginseng saponin R1 in Panax notoginseng
[0026] (1) DNA extraction and high-throughput sequencing:
[0027] 162 natural population materials of Panax notoginseng were collected, genomic DNA was extracted using the CTAB method, and 10X whole genome resequencing was performed.
[0028] (2) Determination of notoginsenoside R1 content:
[0029] A sample of Panax notoginseng was dried at 50°C to a constant weight, crushed using a grinder, and passed through a No. 4 sieve for later use. Using an analytical balance, 0.1 g of Panax notoginseng powder was accurately weighed in 10 mL of methanol. Ultrasonic extraction was performed for 1 hour, and the weight was determined. The weight loss was made up with methanol, and the mixture was shaken thoroughly. The supernatant was filtered through a 0.22 μm organic membrane to obtain the test solution. Notoginsenoside R1 was determined using high-performance liquid chromatography (HPLC). Notoginsenoside R1 was quantitatively analyzed using an external standard method using an Agilent Technologies 1260 HPLC system. The HPLC column was a WelChrom C18 (250 mm × 4.6 mm, 5 μm) column. The mobile phase consisted of acetonitrile (A)-water (B) gradient elution (see Table 1). The flow rate was 1.0 mL / min, the detection wavelength was 203 nm, the column temperature was 25°C, and the injection volume was 10 μL. The compound was identified based on its retention time, and the concentration of notoginsenoside R1 was calculated using the detection peak area of the sample and the standard curve.
[0030] Table 1 Gradient elution program
[0031]
[0032] (3) Genome-wide association study (GWAS)
[0033] Using SNP sites as genotype data and notoginsenoside R1 content as phenotypic data, we imported them into BLINKC (Version 1.01) and performed GWAS analysis using the "BLINK" method (parameter: --gwas). The results are as follows: Figure 1 As shown. Using -log10(P)>6 as the threshold, the SNP molecular marker SNP 5-189765834 that is significantly associated with notoginseng saponin R1 is located at base 189765834 on chromosome Chr5. The reference genome of Panax notoginseng was compared (the reference genome website is: http: / / medicinalplants.ynau.edu.cn / genome), the sequence allelic variation of SNP was extracted, and the notoginseng saponin R1 content of the population material was combined for joint analysis. SNP 5-189765834 has three genotypes: TT, CC, and TC. After performing one-way analysis of variance (non-parametric) using GraphpadPrism 9.5, it was found that the notoginseng saponin R1 content in the CC genotype was higher, which was the dominant genotype ( Figure 2 ).
[0034] Example 2 Development of SNP marker KASP specific primers
[0035] Primers designed using Primer software based on the upstream and downstream sequences of the SNP site (SEQ ID No. 1) were sent to Beijing Qingke Biosynthesis. Each primer mix consists of three primers: two competitive primers and one universal primer. The 3'-terminal bases of the two competitive primers are the variant bases of the SNP site, and the 5' ends are labeled with FAM and HEX fluorescent sequences, respectively. The primer powder was diluted to 10 μM.
[0036] The primer set includes: KASP primers including competitive primers SNP5-FAM and SNP5-HEX for detecting the SNP 5-189765834, and a universal primer SNP5-COM, wherein SNP5-FAM and SNP5-HEX respectively contain FAM and HEX fluorescent linker sequences, and the sequences are as follows:
[0037] SNP5-FAM:
[0038] 5'-GAAGGTGACCAAGTTCATGCTGAACAAAGACAAAGAGACAAAACA-3' (SEQ ID No. 2);
[0039] SNP5-HEX:
[0040] 5'-GAAGGTCGGAGTCAACGGATTGAACAAAGACAAAGAGACAAAACG-3' (SEQ ID No. 3);
[0041] SNP5-COM:
[0042] 5'-TTCTCATAAGTGAATAGCAACCAT-3' (SEQ ID No. 4).
[0043] Example 3 Application Verification of SNP Molecular Markers
[0044] The authenticity of SNP 5-189765834 in a natural population of Panax notoginseng was verified by genotyping using a fluorescent quantitative PCR instrument (Specification: Q2000B, Brand: Langji). Ninety-two individual Panax notoginseng plants were selected, and the notoginsenoside R1 content of each plant was tested using the method of Example 1. Genomic DNA was extracted from each sample, and PCR amplification was performed using the SNP marker KASP-specific primers developed in Example 2 using the genomic DNA as a template.
[0045] The PCR reaction system consisted of 2 μL of DNA template at 40-60 ng / μL; 5 μL of 2× KASP Master Mix; 0.5 μL of KASP primer mix; and 2.5 μL of DEPC water. The upstream primer SNP5-FAM, upstream primer SNP5-HEX, and downstream primer SNP5-COM were mixed in a 1:1:3 volume ratio. The PCR amplification protocol was as follows: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 20 seconds; annealing at 61°C to 55°C for 1 minute, decreasing the temperature by 0.6°C each cycle, for 10 cycles; denaturation at 95°C for 20 seconds, and annealing at 55°C for 1 minute, for 30 cycles. After PCR amplification, the fluorescence signal was read, analyzed, and converted, and the fluorescence scan results were automatically converted into a graph.
[0046] Table 2 Phenotypic content of 92 different genotypes
[0047]
[0048]
[0049] The results in Table 2 were analyzed by one-way ANOVA (nonparametric) using Graphpad Prism 9.5 ( Figure 3 ), which confirmed that the notoginsenoside R1 content in CC genotype was higher, and this SNP molecular marker could be used in genetic improvement of notoginsenoside R1 content in Panax notoginseng.
Claims
1. A method for breeding Panax notoginseng with a high content of notoginseng saponin R1, characterized in that: The genotypes of base 189765834 of chromosome 5 of Panax notoginseng samples were tested, and Panax notoginseng samples with CC genotype at this site were selected as having higher notoginseng saponin R1 content than other genotypes.
2. Application of a substance for detecting the genotype of a SNP site in assisting breeding for the notoginseng saponin R1 content trait. The SNP site is located at position 101 of the nucleotide sequence shown in SEQ ID No. 1 on chromosome 5 of Panax notoginseng. The base of this site has a C / T polymorphism. The genotype of this site is detected and Panax notoginseng with a CC genotype is selected, which has a higher notoginseng saponin R1 content trait than Panax notoginseng with a CT genotype or a TT genotype.
3. The use according to claim 2, characterized in that The substance for detecting the SNP site genotype is a KASP primer set, and the KASP primer set consists of primers shown in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No.
4.
4. The use according to claim 3, characterized in that The method for detecting the genotype of the site is: (1) Extracting genomic DNA from the Panax notoginseng sample to be tested; (2) using the genomic DNA extracted in step (1) as a template and performing PCR amplification using the KASP primer set described in claim 3, (3) Read the fluorescence signal and complete genotyping.
5. The use according to claim 4, characterized in that The PCR amplification procedure was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 20 sec; annealing at 61-55°C for 1 min, with the temperature decreasing by 0.6°C per cycle, for 10 cycles; denaturation at 95°C for 20 sec, and annealing at 55°C for 1 min, for 30 cycles.
6. The use according to claim 4, characterized in that The PCR amplification system is as follows: 2 μL of 40-60 ng / μL DNA template; 5 μL of 2×KASP Master mix; 0.5 μL of KASP primer set, and 2.5 μL of DEPC water; in the KASP primer set, the volume ratio of the competitive primers shown in SEQ ID No. 2 and SEQ ID No. 3 and the universal reverse primer shown in SEQ ID No. 4 is 1:1:
3.
7. KASP primer set, characterized in that The KASP primer set consists of primers shown in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No.
4.
8. A kit, characterized in that Contains the KASP primer set according to claim 7.