SNP site related to content and proportion of ginsenoside Rg1 and Rb1 in panax notoginseng, combination and application of KASP marker

Abstract

The application discloses a SNP site and KASP marker combination related to contents and proportions of ginsenosides Rg1 and Rb1 in Panax notoginseng and application thereof, and belongs to the technical field of medicinal plant molecular breeding and quality evaluation. In view of the problems of lack of molecular markers synchronously related to contents and proportions of ginsenosides Rg1 and Rb1 in Panax notoginseng and long breeding cycle in the prior art, nine SNP site combinations located on a Chr11 chromosome of Panax notoginseng genome are screened, and a KASP marker combination and primer combination are developed based on four sites of SNP3-SNP6 among them. The KASP marker combination has strong specificity, is convenient to detect, has high throughput, is low in cost, and can accurately identify the contents and proportions of Rg1 and Rb1 in Panax notoginseng samples. The application can be used for Panax notoginseng germplasm resource screening and molecular marker assisted breeding, early identification of target traits is realized, the breeding cycle is shortened, the breeding efficiency is improved, and technical support is provided for directional cultivation of Panax notoginseng high-quality varieties.

Description

Technical Field

[0001] This invention relates to the field of molecular marker-assisted breeding technology, and in particular to the combination and application of SNP sites and KASP markers related to the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng. Background Technology

[0002] Panax notoginseng, a perennial herb belonging to the genus Panax in the family Araliaceae, is a precious and unique traditional Chinese medicine. Its dried roots and rhizomes are used medicinally, possessing properties that disperse blood stasis, stop bleeding, reduce swelling, and relieve pain. Modern pharmacological studies have confirmed that Panax notoginseng exhibits significant activity in cardiovascular protection, anti-inflammation, and antioxidant effects. It is a core ingredient in many traditional Chinese medicine preparations, including Yunnan Baiyao, Xue Sai Tong soft capsules, and Compound Danshen Dripping Pills, and market demand continues to grow.

[0003] Ginsenosides are the most important active components of Panax notoginseng, and their content and composition directly determine the quality of the Panax notoginseng. Among them, ginsenosides Rg1 and Rb1 are the two monomeric components with the highest content and most clearly defined pharmacological activities in the total saponins of Panax notoginseng. Studies have shown that Rg1 has effects such as improving cognitive function and anti-inflammation, while Rb1 has a protective effect on vascular endothelium. The two work synergistically to promote blood circulation, remove blood stasis, and protect the cardiovascular and cerebrovascular systems. Therefore, the content and ratio of ginsenosides Rg1 and Rb1 are key indicators for evaluating the quality of Panax notoginseng. Targeted cultivation of Panax notoginseng varieties with high Rg1 and Rb1 content and appropriate ratios is of great significance for improving the quality of Panax notoginseng.

[0004] Currently, the breeding of Panax notoginseng varieties mainly adopts the group mixed selection method, relying on phenotypic selection. Panax notoginseng is a perennial plant, and each growth generation requires at least 3 years. Moreover, there is a lack of effective early selection and identification methods, resulting in a breeding cycle of 15 to 20 years and extremely low efficiency. At the same time, the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng are affected by both genetic and environmental factors. Relying solely on phenotypic selection is insufficient to accurately screen out genetically stable and superior germplasm.

[0005] Molecular marker-assisted breeding (MAS) can overcome the limitations of phenotypic selection and achieve early and precise screening of target traits. Single nucleotide polymorphism (SNP) markers have become commonly used markers in molecular breeding due to their wide distribution, high density, and convenient detection. Competitive allele-specific PCR (KASP) technology, based on SNP sites, has advantages such as low cost, simple operation, high throughput, and accurate results, making it suitable for large-scale germplasm screening. However, current technologies lack SNP site combinations and corresponding KASP markers that can simultaneously correlate the content and ratio of ginsenosides Rg1 and Rb1, making it impossible to achieve simultaneous and precise detection and early screening of the content and ratio of Rg1 and Rb1 in Panax notoginseng.

[0006] Therefore, identifying SNP sites that are significantly related to the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng, developing corresponding KASP marker combinations, and establishing efficient detection methods are of great significance for shortening the breeding cycle of Panax notoginseng, improving breeding efficiency, and promoting the high-quality development of the Panax notoginseng industry. Summary of the Invention

[0007] The purpose of this invention is to provide a combination of SNP sites and KASP markers related to the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng, and their application, in order to solve the above-mentioned technical problems.

[0008] The above-mentioned technical objective of the present invention is achieved through the following technical solution: A combination of SNP sites related to the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng, wherein the combination of SNP sites is located on chromosome Chr11 of the Panax notoginseng genome and consists of the following 9 SNP sites: SNP1 is located at position 131495227 on chromosome 11 and is mutated to C / T. SNP2 is located at position 131495511 on chromosome 11 and is mutated to C / T. SNP3 is located at position 131496703 on chromosome 11 and is mutated to C / T. SNP4 is located at position 131497774 on chromosome 11 and is mutated to T / G. SNP5 is located at position 131498364 on chromosome 11 and is mutated to C / T. SNP6 is located at position 131498672 on chromosome 11 and is mutated to T / C. SNP7 is located at position 131498680 on chromosome 11 and is mutated to G / A. SNP8 is located at position 131500483 on chromosome 11 and is mutated to T / A. SNP9 is located at position 131501173 on chromosome 11 and is mutated to A / T.

[0009] The present invention also provides a KASP marker combination related to the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng. The KASP marker combination is used to detect the four SNP sites SNP3, SNP4, SNP5 and SNP6 mentioned above. The nucleotide sequences of the KASP marker combination are shown in SEQ ID NO.1-4, respectively.

[0010] The present invention also provides a KASP marker combination related to the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng. The KASP primer combination is used to detect the above-mentioned four SNP sites SNP3, SNP4, SNP5 and SNP6, and their nucleotide sequences are shown in SEQ ID NO.5-16, respectively.

[0011] The present invention also provides a kit comprising the KASP primer combination as described in claim 3.

[0012] More preferably, the kit is used for the detection of the content and proportion of ginsenosides Rg1 and Rb1 in Panax notoginseng.

[0013] This invention also provides the application of the above-mentioned SNP site combinations, KASP marker combinations, KASP primer combinations, or kits in the identification of the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng or in molecular-assisted breeding.

[0014] This invention also provides a method for detecting the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng, comprising the following steps: S1. Extract genomic DNA from the Panax notoginseng sample to be tested; S2. Using the genomic DNA obtained in step S1 as a template, perform PCR amplification using the KASP primer combination described in claim 3. S3. Detect the fluorescence signal of the PCR amplification product, determine the genotype of each SNP site, and judge the content and ratio of ginsenosides Rg1 and Rb1 in the Panax notoginseng sample based on the genotype detection results.

[0015] Further preferably, the PCR amplification reaction procedure in step S2 includes: first, pre-denaturation at 95°C for 10 minutes; then denaturation at 95°C for 20 seconds; followed by 10 cycles of falling PCR annealing, with the annealing temperature decreasing by 0.6°C per cycle from 61°C to 55°C for 40 seconds; and finally, 40 cycles of conventional PCR, each cycle including denaturation at 95°C for 20 seconds and annealing at 55°C for 40 seconds.

[0016] More preferably, the PCR amplification reaction system in step S2 comprises: 1 μL of 2× KASP Master Mix, 1 μL of template DNA with a concentration of 10-50 ng / μL, and 0.01 μL of the KASP primer combination as described in claim 3, wherein the volume ratio of SNP-F, SNP-H, and SNP-R is 1:1:3.

[0017] More preferably, step S3, which involves determining the content and ratio of ginsenosides Rg1 and Rb1 in the Panax notoginseng sample based on genotype detection results, specifically involves: The content of ginsenoside Rg1 and the Rg1 / Rb1 ratio in Panax notoginseng samples with genotype combinations of TT / GG / TT / CC were higher than those with genotype combinations of CT / TG / CT / TC or CC / TT / CC / TT. The content of ginsenoside Rb1 in Panax notoginseng samples with the genotype combination TT / GG / TT / CC was lower than that in Panax notoginseng samples with the genotype combination CT / TG / CT / TC or CC / TT / CC / TT. The genotype combinations are arranged in the order of SNP3 / SNP4 / SNP5 / SNP6.

[0018] In summary, the present invention has the following beneficial effects: Firstly, this invention provides for the first time a combination of 9 SNP sites that are significantly associated with the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng. This combination was obtained from 271 natural populations of Panax notoginseng through genome-wide association analysis (GWAS), filling the gap in the existing technology of lacking SNP sites that synchronously associate the content and ratio of Rg1 and Rb1.

[0019] Secondly, this invention developed four sets of KASP primer combinations based on four key sites, SNP3-SNP6, which can specifically distinguish the bases at mutation sites and achieve accurate detection of the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng.

[0020] Thirdly, the KASP marker combination provided by this invention has the advantages of high specificity, convenient detection, high throughput and low cost. It can be applied to the screening of Panax notoginseng germplasm resources and molecular marker-assisted breeding, realize the early identification of target traits, effectively shorten the breeding cycle, improve breeding efficiency, and provide reliable technical support for the targeted breeding of high-quality Panax notoginseng varieties. Attached Figure Description

[0021] Figure 1 Manhattan and QQ-plots of GWAS results for the contents of ginsenosides Rg1 and Rb1 in Panax notoginseng; Figure 2 A statistical graph showing the allelic variation and phenotypic significance of the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng; Figure 3 Genotyping results of different Panax notoginseng samples using KASP-specific primers. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Example 1: Obtaining nucleotide mutation sites (SNPs) related to the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng. (1) DNA extraction and high-throughput sequencing: We collected 271 Panax notoginseng natural population materials, extracted genomic DNA using the CTAB method, and performed 10X whole-genome resequencing.

[0024] (2) Determination of ginsenoside Rg1 and Rb1 content: After drying at 50℃, the sample was pulverized using a pulverizer and passed through a No. 4 sieve. 0.6 g of the sample was accurately weighed using an analytical balance and placed in a 50 ml Erlenmeyer flask. 50 ml of methanol was added, and the flask was weighed. The flask was sealed with sealing film, sonicated for 30 min, and then allowed to stand for 20 h. The sealing film was then removed, and the sample was weighed again. The weight lost due to evaporation was replenished with methanol, and the mixture was shaken well. The solution was filtered through a 0.22 μm microporous membrane to obtain 1 ml of the sample solution. Ginsenosides Rg1 and Rb1 were quantitatively analyzed using high-performance liquid chromatography (HPLC) with the external standard method. The chromatographic column was an Agilent ZORBAX SB-AQ (3.5µm, 4.6×150mm). The mobile phase consisted of ultrapure water (A) and acetonitrile (B). The gradient elution program was as follows: 0–20 min, 20% (B); 20–55 min, 36% (B); 55–60 min, 40% (B); 60–70 min, 45% (B); 70–79 min, 60% (B); 79–80 min, 80% (B); 80–81.5 min, 80% (B); 81.5–83 min, 20% (B). The flow rate was 0.5 ml / min, the column temperature was 30℃, and the injection volume was 10 µl. The detection wavelength was 203 nm. The compounds were identified based on their retention time, and quantification was performed using the external standard method, with peak area as the quantification basis.

[0025] (3) Genome-wide association analysis (GWAS) Using SNP loci as genotypic data and the contents of ginsenosides Rg1 and Rb1 as phenotypic data, genome-wide association studies (GWAS) were performed using EMMAX software with a mixed linear model (MLM). Manhattan plots and QQ-plots were obtained, as shown below. Figure 1 As shown. Using -log 10(P)>6 was used as the threshold. In the Manhattan plot, the X-axis represents each SNP on all chromosomes, and the Y-axis represents the P-value of each SNP. Different colors represent the 12 chromosomes of Panax notoginseng. SNPs above the horizontal line were selected as candidate significant SNP sites. The QQ-plot can infer the rationality of the model and the location of SNP sites by comparing the positions of predicted and observed values. Among them, 9 SNP sites (SNP1-9) that were significantly associated with the content of ginsenosides Rg1 and Rb1 were located on chromosome 11. As shown in Table 1. The sequence allelic variation of SNP3, SNP4, SNP5, and SNP6 was extracted and analyzed in combination with the content of ginsenosides Rg1 and Rb1 in the population material. The ratio of ginsenoside Rg1 and Rg1 / Rb1 content in Panax notoginseng with the genotype combination TT / GG / TT / CC was higher than that in Panax notoginseng with the genotype combination CT / TG / CT / TC or CC / TT / CC / TT. Rb1 showed the opposite trend. Figure 2 As shown.

[0026] Table 1. Nine SNP sites significantly associated with the contents of both ginsenoside Rg1 and Rb1. The molecular marker nucleotide sequences are shown in SEQ ID NO.1-4.

[0027] Example 2: Development of SNP-labeled KASP-specific primers Based on the above 9 SNP sites, 4 KASP molecular markers for KASP detection were developed. Using NCBI's Primer-BLAST function, three primers were designed based on the sequence of SEQ ID NO.1: upstream primer SNP-F, upstream primer SNP-H, and downstream primer SNP-F. SNP-F and SNP-H contain FAM and HEX fluorescent linker sequences (underlined), respectively. The sequences are as follows: Table 2 KASP primer sequences Example 3: Genotyping of SNP loci in different Panax notoginseng samples and its application The authenticity of the four KASP molecular markers in the natural population of Panax notoginseng was verified using the high-throughput genotyping system GeneMatrix (GM). Genomic DNA was extracted from 94 randomly selected Panax notoginseng single plants. Using the genomic DNA as a template, PCR amplification was performed using the SNP marker KASP-specific primers developed in Example 2.

[0028] After diluting the sample DNA 5-fold, transfer it to a 100 μL PCR plate, and add 2 positive controls and 2 negative controls (NTC) to each plate.

[0029] The KASP reaction system consisted of 2 μL, including 1 μL of 2×Master Mix, 1 μL of 20 ng / μL sample DNA, and 0.01 μL of KASP primer mixture, comprising 0.002 μL of upstream primer SNP-F (100 μM), 0.002 μL of upstream primer SNP-H (100 μM), and 0.006 μL of downstream primer SNP-R (100 μM). An equal volume of double-distilled water was used instead of sample DNA in the negative control reaction. The DNA sample plate, primer mixture, and KASP master mix were placed in the corresponding positions on the Arrayer. The 384*1 pipetting protocol was selected, and the DNA and primer mixture were added to the 384-well microplate via the Matrix Arrayer. The device automatically constructed the reaction system and heat-sealed the reaction plate.

[0030] The PCR amplification program requires four stages: Stage 1 is pre-denaturation at 95℃ for 10 min; Stage 2 is continuous denaturation at 95℃ for 20 s and annealing at 61℃ for 40 s, for a total of 10 cycles (each cycle decreasing by 0.6℃); Stage 3 is denaturation at 95℃ for 20 s and annealing at 55℃ for 40 s, for a total of 40 cycles.

[0031] After PCR amplification, remove the reaction plate and allow it to cool to room temperature before placing it in a scanner for fluorescence scanning. Use a Matrix Scanner to read the fluorescence signal values ​​and perform genotyping and clustering of the samples. Samples displayed in blue near the Y-axis represent alleles linked to the HEX fluorescent tag sequence; samples displayed in red near the X-axis represent alleles linked to the FAM fluorescent tag sequence. Figure 3 As shown.

[0032] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A combination of SNP sites related to the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng, characterized in that, The SNP locus combination is located on chromosome Chr11 of the Panax notoginseng genome and consists of the following 9 SNP loci: SNP1 is located at position 131495227 on chromosome 11 and is mutated to C / T. SNP2 is located at position 131495511 on chromosome 11 and is mutated to C / T. SNP3 is located at position 131496703 on chromosome 11 and is mutated to C / T. SNP4 is located at position 131497774 on chromosome 11 and is mutated to T / G. SNP5 is located at position 131498364 on chromosome 11 and is mutated to C / T. SNP6 is located at position 131498672 on chromosome 11 and is mutated to T / C. SNP7 is located at position 131498680 on chromosome 11 and is mutated to G / A. SNP8 is located at position 131500483 on chromosome 11 and is mutated to T / A. SNP9 is located at position 131501173 on chromosome 11 and is mutated to A / T.

2. A KASP marker combination related to the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng, characterized in that: The KASP marker combination is used to detect the four SNP sites SNP3, SNP4, SNP5 and SNP6 as described in claim 1, and the nucleotide sequences of the KASP marker combination are shown in SEQ ID NO.1-4, respectively.

3. A KASP marker combination related to the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng, characterized in that: The KASP primer combination is used to detect the four SNP sites SNP3, SNP4, SNP5 and SNP6 as described in claim 1, and their nucleotide sequences are shown in SEQ ID NO.5-16, respectively.

4. A reagent kit, characterized in that, The kit comprises the KASP primer combination as described in claim 3.

5. The reagent kit according to claim 4, characterized in that, The kit is used to detect the content and proportion of ginsenosides Rg1 and Rb1 in Panax notoginseng.

6. The application of the SNP site combination of claim 1, the KASP marker combination of claim 2, the KASP primer combination of claim 3, or the kit of claim 4 in the identification of the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng or in molecular-assisted breeding.

7. A method for detecting the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng, characterized in that, Includes the following steps: S1. Extract genomic DNA from the Panax notoginseng sample to be tested; S2. Using the genomic DNA obtained in step S1 as a template, perform PCR amplification using the KASP primer combination described in claim 3. S3. Detect the fluorescence signal of the PCR amplification product, determine the genotype of each SNP site, and judge the content and ratio of ginsenosides Rg1 and Rb1 in the Panax notoginseng sample based on the genotype detection results.

8. The method for detecting the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng according to claim 7, characterized in that: The PCR amplification reaction procedure described in step S2 includes: first, pre-denaturation at 95°C for 10 minutes; then denaturation at 95°C for 20 seconds; followed by 10 cycles of falling PCR annealing, with the annealing temperature decreasing by 0.6°C from 61°C to 55°C for 40 seconds each cycle; and finally, 40 cycles of conventional PCR, each cycle including denaturation at 95°C for 20 seconds and annealing at 55°C for 40 seconds.

9. The method for detecting the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng according to claim 7, characterized in that: The PCR amplification reaction system described in step S2 comprises: 1 μL of 2× KASP Master Mix, 1 μL of template DNA with a concentration of 10-50 ng / μL, and 0.01 μL of the KASP primer combination as described in claim 3, wherein the volume ratio of SNP-F, SNP-H, and SNP-R is 1:1:

3.

10. The method for detecting the content and ratio of ginsenosides Rg1 and Rb1 in Panax notoginseng according to claim 7, characterized in that: Step S3, which involves determining the content and ratio of ginsenosides Rg1 and Rb1 in the Panax notoginseng sample based on genotype detection results, specifically involves: The content of ginsenoside Rg1 and the Rg1 / Rb1 ratio in Panax notoginseng samples with genotype combinations of TT / GG / TT / CC were higher than those with genotype combinations of CT / TG / CT / TC or CC / TT / CC / TT. The content of ginsenoside Rb1 in Panax notoginseng samples with the genotype combination TT / GG / TT / CC was lower than that in Panax notoginseng samples with the genotype combination CT / TG / CT / TC or CC / TT / CC / TT. The genotype combinations are arranged in the order of SNP3 / SNP4 / SNP5 / SNP6.

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