SNP (Single Nucleotide Polymorphism) molecular marker related to content of ginsenoside Re in panax notoginseng and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to the content of ginsenoside Re in panax notoginseng and application, the SNP molecular marker is SNP10-13886209 and is located at the 13886209th basic group of Chr10 chromosome, and the mutation type of the SNP molecular marker is G / T. The KASP primer combination developed by the invention can accurately distinguish pseudo-ginseng with high ginsenoside Re content from pseudo-ginseng with low ginsenoside Re content, can be applied to pseudo-ginseng molecular-assisted marker breeding and shorten the breeding period of new varieties, and has the advantages of low detection cost, no environmental limitation, high detection result accuracy and easy repetition. The method has important theoretical and practical guiding significance for accelerating the genetic improvement process of panax notoginseng breeding with high ginsenoside Re content and improving the breeding selection efficiency.

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 ginsenoside Re 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 variety selection. 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. Panax notoginseng variety selection still relies on traditional systematic breeding. However, each generation of Panax notoginseng requires at least three years, and the speed of phenotypic separation and population construction is extremely slow. There is also a lack of early selection and identification methods, 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 precise association analysis between markers and traits, and improve the efficiency of MAS breeding. Ginsenoside Re is a characteristic component of Panax notoginseng saponins. Its content in Panax notoginseng is a key indicator of its quality and, therefore, a core objective in cultivar selection. Therefore, leveraging known saponin biosynthesis genes and the abundant Panax notoginseng germplasm resources, it is crucial to identify SNPs significantly associated with ginsenoside Re 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 ginsenoside Re in Panax notoginseng and its application.

[0007] The technical solution of the present invention is: the use of a substance for detecting SNP site polymorphism or genotype in the Panax notoginseng genome in any of the following:

[0008] (1) Identify or assist in identifying the content of ginsenoside Re in Panax notoginseng;

[0009] (2) Breeding or assisting in the breeding of Panax notoginseng lines with high ginsenoside Re content;

[0010] (3) preparing products for identifying or assisting in identifying the content of ginsenoside Re in Panax notoginseng;

[0011] (4) preparing products for breeding or assisting in breeding Panax notoginseng strains with high ginsenoside Re content;

[0012] The SNP site is located at position 301 of the nucleotide sequence shown in SEQ ID No. 1 on chromosome 10 of Panax notoginseng. The site has a T / G polymorphism. The genotype of the site is detected and Panax notoginseng with a genotype of TT is selected, which has a higher ginsenoside Re content trait than Panax notoginseng with a genotype of TG or GG.

[0013] Furthermore, the substance 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.

[0014] Furthermore, the method for detecting the genotype of the site is:

[0015] (1) Extracting genomic DNA from the Panax notoginseng sample to be tested;

[0016] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the KASP primer set described above.

[0017] (3) Read the fluorescence signal and complete genotyping.

[0018] 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.

[0019] Furthermore, the PCR amplification system is: 2 μL of 40-60 ng / μL DNA template, 5 μL of 2×KASP Mastermix, 2.5 μL of DEPC water, and 0.5 μL of KASP primer set; 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.

[0020] 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.

[0021] A kit comprising the above-mentioned KASP primer set.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a single nucleotide polymorphism (SNP) significantly associated with ginsenoside Re content in Panax notoginseng. Association analysis was conducted on 162 natural Panax notoginseng populations. Using the SNP loci as genotype data and ginsenoside Re 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 SNP10-13886209, located at base 13886209 on chromosome Chr10, provides technical support for molecular marker-assisted breeding for the ginsenoside Re content trait in Panax notoginseng.

[0024] The KASP primer combination developed in the present invention can directly and specifically distinguish and detect the G or T base at the mutation site of SNP10-13886209. When used to identify high and low ginsenoside Re content, the KASP primer combination can clearly separate the two genotypes. The KASP primer combination developed in the present invention has excellent application value and can enable pre-selection and molecular-assisted breeding for the ginsenoside Re content trait in Panax notoginseng. This has important theoretical and practical guiding significance for the genetic improvement of ginsenoside Re content in Panax notoginseng and improving breeding selection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Manhattan and QQ-plot of GWAS results of ginsenoside Re in Panax notoginseng;

[0026] Figure 2 This is a statistical diagram of the allelic variation and phenotypic significance of ginsenoside Re content in Panax notoginseng; *p<0.05, ***p<0.001;

[0027] Figure 3 This is a statistical diagram of the allelic variation and phenotypic significance of ginsenoside Re content in 92 Panax notoginseng samples; among them, *p<0.05, ***p<0.001. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1 Obtaining Nucleotide Mutation Sites (SNPs) Related to Ginsenoside Re Content in Panax Notoginseng

[0030] (1) DNA extraction and high-throughput sequencing:

[0031] 162 natural population materials of Panax notoginseng were collected, genomic DNA was extracted using the CTAB method, and 10X whole genome resequencing was performed.

[0032] (2) Determination of ginsenoside Re content:

[0033] A sample of Panax notoginseng was dried at 50°C to a constant weight, crushed in 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. Ginsenoside Re was quantitatively analyzed using high-performance liquid chromatography (HPLC) using an external standard method. The HPLC was performed on an Agilent Technologies 1260 instrument with a WelChrom C18 column (250 mm × 4.6 mm, 5 μm). 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 compounds were identified based on their retention times, and the concentration of ginsenoside Re was calculated using the detection peak area of ​​the samples and the standard curve.

[0034] Table 1 Gradient elution program

[0035]

[0036] (3) Genome-wide association study (GWAS)

[0037] Using SNP sites as genotype data and ginsenoside Re 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)>5 as the threshold, the SNP molecular marker SNP10-13886209, which is significantly associated with ginsenoside Re, is located at base 13886209 on chromosome Chr10. The reference genome of Panax notoginseng (reference genome website: http: / / medicinalplants.ynau.edu.cn / genome) was compared to extract the sequence allele variation of SNPs. Combined with the ginsenoside Re content of the population materials, a joint analysis was performed. SNP10-13886209 has three genotypes: GG, TT, and GT. After a one-way analysis of variance (non-parametric) using GraphpadPrism 9.5, it was found that the ginsenoside Re content in the TT genotype was higher, making it the dominant genotype ( Figure 2 ).

[0038] Example 2 Development of SNP marker KASP specific primers

[0039] 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.

[0040] The primer set includes: KASP primers including competitive primers SNP10-FAM and SNP10-HEX for detecting the SNP10-13886209, and a universal primer SNP10-COM, wherein SNP10-FAM and SNP10-HEX respectively contain FAM and HEX fluorescent linker sequences, and the sequences are as follows:.

[0041] SNP10-FAM: 5'-GCCGGTGACCCGTTCATGCTATGACACCCAAGCCAAAACAG-3' (SEQ IDNo. ​​2);

[0042] SNP10-HEX: 5'-GCCGGTCGGAGTCCCCGGATTATGACACCCAAGCCAAAACAT-3 (SEQ IDNo. ​​3);

[0043] SNP10-COM: 5'-CGGGCTAAAGAACGACACC-3' (SEQ ID No. 4).

[0044] Example 3 Detection of genotypes of SNP sites in different Panax notoginseng samples and their applications

[0045] Genotyping of SNP 10-13886209 in a natural population of Panax notoginseng was verified using a fluorescent quantitative PCR instrument (Q2000B, brand: Langji). Ninety-two individual Panax notoginseng plants were selected, and the ginsenoside Re content of each plant was assayed 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.

[0046] 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 competitive primers SNP10-FAM and SNP10-HEX, and the universal primer SNP10-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.

[0047] Table 2 Phenotypic content of different genotypes

[0048]

[0049]

[0050] The results in Table 2 were analyzed by one-way ANOVA (nonparametric) using Graphpad Prism 9.5 ( Figure 3 ), which confirmed that the TT genotype had a higher content of ginsenoside Re, and this SNP molecular marker could be used in the genetic improvement of notoginseng saponin Re content in Panax notoginseng.

Claims

1. Application of a substance for detecting SNP polymorphism or genotype in the genome of Panax notoginseng in any of the following: (1) Identify or assist in identifying the content of ginsenoside Re in Panax notoginseng; (2) Breeding or assisting in breeding Panax notoginseng lines with high ginsenoside Re content; (3) preparing products for identifying or assisting in identifying the content of ginsenoside Re in Panax notoginseng; (4) preparing products for breeding or assisting in breeding Panax notoginseng strains with high ginsenoside Re content; The SNP site is located at position 301 of the nucleotide sequence shown in SEQ ID No. 1 on chromosome 10 of Panax notoginseng. The base of this site has a T / G polymorphism. The genotype of this site is detected and Panax notoginseng with a genotype of TT is selected, which has a higher ginsenoside Re content trait than Panax notoginseng with a genotype of TG or GG.

2. The use according to claim 1, characterized in that The substance 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.

3. The use according to claim 2, 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 2, (3) Read the fluorescence signal and complete genotyping.

4. The use according to claim 3, 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.

5. 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, 2.5 μL of DEPC water, and 0.5 μL of KASP primer set; 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.

6. 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.

7. A kit, characterized in that Contains the KASP primer set according to claim 6.

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