SNP (Single Nucleotide Polymorphism) molecular marker combination for identifying new variety of Scutellaria baicalensis Guoqin1 and construction method and application of molecular identity card

Abstract

The invention relates to the technical field of molecular biology, in particular to an SNP (Single Nucleotide Polymorphism) molecular marker combination for identifying a new variety of radix scutellariae, namely Guoqin No.1, and a construction method and application of a molecular identity card. According to the invention, the SNP molecular marker combination for identifying a new variety of radix scutellariae Guoqin No.1 is determined by utilizing an SNP molecular marker technology, and a molecular identity card of the Guoqin No.1 is constructed, so that the application of the molecular identity card in identifying the varieties of traditional Chinese medicinal materials is widened, a foundation is laid for identifying the varieties of other traditional Chinese medicinal materials and establishing a provenance traceability system by the molecular identity card, and the application prospect of the molecular identity card in identifying the varieties of other traditional Chinese medicinal materials is widened. And a theoretical support is provided for establishing a variety identification standard.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and specifically relates to a method and application for constructing and identifying SNP molecular marker combinations and molecular identity cards for the new Scutellaria baicalensis variety Guoqin No. 1. Background Technology

[0002] Scutellaria baicalensis, a major medicinal herb, possesses therapeutic effects such as clearing heat and drying dampness, purging fire and detoxifying, and stopping bleeding. In December 2022, Guoqin No. 1 was granted plant variety rights by the National Forestry and Grassland Administration, becoming the first Scutellaria baicalensis plant variety to receive such rights nationwide. However, identification methods for this germplasm are currently lacking. While traditional traits and physiological and biochemical indicators can identify medicinal herb germplasm resources to some extent, they are insufficient for accurate identification when faced with germplasm resources of the same species from different sources, as well as closely related similar varieties and adulterants. With the continuous deepening of the breeding of new medicinal herb varieties, accurate identification of new medicinal herb varieties with mainstream cultivated varieties and wild resources is more conducive to the healthy development of the medicinal herb industry. Summary of the Invention

[0003] To address the aforementioned problems, in a first aspect, this invention discloses a combination of SNP molecular markers for identifying the new Scutellaria baicalensis variety, Guoqin No. 1, wherein the combination of SNP molecular markers consists of nine SNP sites, SNP1-SNP9: SNP1 is located at physical position 3566028 on chromosome Sbai1, in gene Sbai1A35T79, and its polymorphism is T / A. SNP2 is located at physical position 1872824 on chromosome Sbai4, and the gene is Sbai4C18T15. Its polymorphism is A / T. SNP3 is located at physical position 2351968 on chromosome Sbai4, in gene Sbai4A23T90, and its polymorphism is A / G; SNP4 is located at physical position 8349245 on chromosome Sbai4, and the gene is Sbai4C83T14. Its polymorphism is C / T. SNP5 is located at physical position 9793077 on chromosome Sbai4, in gene Sbai4A98T73, and its polymorphism is C / T. SNP6 is located at physical position 18269593 on chromosome Sbai4, and the gene is Sbai4C182T8, with a polymorphism of C / T. SNP7 is located at physical position 22292483 on chromosome Sbai4, and the gene is Sbai4A223T75. Its polymorphism is G / A. SNP8 is located at physical position 25585245 on chromosome Sbai4, and the gene is Sbai4P255T65. Its polymorphism is G / C. SNP9 is located at physical position 37711547 on chromosome Sbai4, in gene Sbai4C377T4, and its polymorphism is C / T.

[0004] Furthermore, the nucleotide sequences of the nine SNP sites from SNP1 to SNP9 are shown in SEQ ID NO.1 to SEQ ID NO.9, respectively; The bases of the nine SNP sites from SNP1 to SNP9 correspond to the 101st position of the sequences shown in SEQ ID NO.1 to SEQ ID NO.9.

[0005] Secondly, this invention proposes a method for obtaining and identifying SNP molecular marker combinations for the new Scutellaria baicalensis variety, Guoqin No. 1, comprising the following steps: Obtain Scutellaria baicalensis samples, including Guoqin No. 1, and extract total DNA from each Scutellaria baicalensis sample; The extracted total DNA was subjected to quality testing. Sequencing libraries were constructed using DNA samples that met the quality standards, and the quality of the libraries was verified. The validated libraries were subjected to high-throughput sequencing and quality control processing to obtain DNA quality control sequences. The DNA quality control sequence was compared with the Scutellaria baicalensis reference genome GWHDEDD00000000 to determine the nine SNP molecular site marker combinations from SNP1 to SNP9.

[0006] Furthermore, a qualified DNA sample must meet at least the following conditions: The OD260 / 280 ratio for nucleic acid purity testing of DNA is between 1.8 and 2.2. The nucleic acid concentration of the DNA is not less than 50 ng / μL, and the total content is not less than 2 μg.

[0007] Furthermore, the quality control process includes: The high-throughput sequencing data were filtered to ensure that the proportion of Q30 bases was not less than 90%.

[0008] Thirdly, this invention proposes a method for constructing the molecular identity card of a new Scutellaria baicalensis variety, Guoqin No. 1, comprising the following steps: The nine SNP molecular marker combinations of SNP1-SNP9 were obtained according to the method used to obtain and identify the new Scutellaria baicalensis variety Guoqin No. 1. The genotypes of the nine SNP loci (SNP1-SNP9) were sequentially coded digitally to obtain the molecular identification of Guoqin No. 1.

[0009] Furthermore, the genotype order of the nine SNP loci from SNP1 to SNP9 is AA, TT, GG, CC, TT, TT, AA, GG, TT.

[0010] Furthermore, the step of sequentially digitally encoding the genotypes of the nine SNP loci (SNP1-SNP9) in Guoqin No. 1 includes: The bases of the nine SNP sites (SNP1-SNP9) in Guoqin No. 1 were digitally encoded using the numbers 0-4, where A=1, T=2, G=3, C=4, and N=0. The molecular identity card of Guoqin No. 1 is generated by sequentially cascading the digital codes of the nine SNP sites from SNP1 to SNP9.

[0011] Fourthly, this invention proposes a molecular identity card for a new Scutellaria baicalensis variety, Guoqin No. 1, which is constructed by a method for constructing the molecular identity card for the new Scutellaria baicalensis variety, Guoqin No. 1.

[0012] Fifthly, this invention proposes a method for identifying a new Scutellaria baicalensis variety, Guoqin No. 1, the method comprising the following steps: Obtain the genotype of the sample to be identified. If the genotype of the sample to be identified is consistent with the genotype of the 9 SNP loci from SNP1 to SNP9, it is identified as the new Scutellaria baicalensis variety Guoqin No. 1. Alternatively, the method may include the following steps: Obtain the molecular identification of the sample to be identified. If the molecular identification of the sample to be identified is consistent with that of Guoqin No. 1, it is determined to be the new Scutellaria baicalensis variety Guoqin No. 1.

[0013] The beneficial effects of this invention are: This invention utilizes SNP molecular marker technology to construct a molecular identification system for the new Scutellaria baicalensis variety, Guoqin No. 1. This system enables rapid and accurate identification of the authenticity of the parent plants and their offspring seeds, protecting plant variety rights, preventing counterfeit and substandard varieties from entering the market, and ensuring the safety and reliability of superior seed sources. It also broadens the application of molecular identification systems in the identification of Chinese medicinal herbs, laying the foundation for the establishment of identification and seed source traceability systems for other Chinese medicinal herbs, and providing theoretical support for establishing variety identification standards. Furthermore, it ensures the safety and reliability of the introduction of Guoqin No. 1, providing an important guarantee for the high-quality development of the Scutellaria baicalensis industry.

[0014] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures pointed out in the description and claims. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1 Screening of SNP locus combinations for the new Scutellaria baicalensis variety, Guoqin No. 1: Step 1: Collect a total of 107 tissue samples of Scutellaria baicalensis, including Guoqin No. 1; Step 2: Total DNA extraction, quality testing, and genotyping; 2.1 DNA was extracted from the samples using a universal genomic DNA extraction kit (magnetic bead method, China Migi-Yuhua kit) to obtain total DNA from the Scutellaria baicalensis samples; 2.2 The total DNA of the Scutellaria baicalensis samples was detected by 1% agarose gel electrophoresis using a DYY-6C electrophoresis system. 2.3 The total DNA concentration of Scutellaria baicalensis samples was detected using a Nanodrop UV spectrophotometer. The retention absorbance ratio (OD260 / 280) was between 1.8 and 2.2. The concentration was greater than 50 ng / μL and the total amount was greater than 2 μg using a Qubit 3.0 spectrophotometer.

[0017] 2.4 After the DNA samples passed the initial testing, they were randomly fragmented using a Covaris ultrasonic disruptor. The entire library preparation process then involved end repair, A-tailing, sequencing adapter addition, purification, and PCR amplification. After library construction, preliminary quantification was performed using Qubit 3.0 to dilute the library. Subsequently, the insert fragments were detected using an Agilent 2100. Once the insert fragment size met expectations, the effective concentration of the library was accurately quantified using Q-PCR to ensure library quality.

[0018] 2.5 After the libraries pass the detection, different libraries are pooled into flow cells according to the effective concentration and the required amount of data to be sequenced. After cBOT clustering, sequencing is performed using the Illumina NovaSeq X plus high-throughput sequencing platform.

[0019] Step 3: Sequencing data quality control and sequence alignment; 3.1 Raw sequencing data (FASTQ format) was filtered using Trimmomatic, low-quality bases (HEADCROP: 10) were removed, and adapter contamination was eliminated to generate high-quality Clean Reads. Data quality was assessed using FastQC, including base quality distribution, GC content, and sequence reproducibility, to ensure that the Q30 base ratio was ≥90% and there was no excessive shift or contamination.

[0020] 3.2 The BWA (v0.7.13-r1126) software was used to align the quality-controlled sequences to the reference genome GWHDEDD00000000 (accessed by the National Center for Biotechnology Information, accession number GWHDEDD00000000) with the default parameters. Based on the alignment results, the genotype of the target site was calculated using samtools software (version 0.1.18). Finally, Annovar software was used to annotate the mutation sites.

[0021] Step 4: SNP site screening; 4.1 A total of 1,121,566 original SNP loci were obtained through raw data analysis. To ensure the accuracy and reliability of subsequent analysis results, all SNP loci underwent rigorous quality filtering, deletion filtering, and minor allele frequency filtering. Additionally, dialexical and 4DTv loci were also screened. After multiple rounds of rigorous screening, 24,783 high-quality SNP loci remained.

[0022] 4.2 Candidate SNPs were filtered and screened from high-quality SNP loci using PLINK software. The specific criteria were as follows: a. biallelic loci; b. 4-fold degenerate SNPs; c. heterozygosity <0.2; d. deletion rate <0.2; e. minor allele frequency (MAF) >0.05; f. no InDel or SNPs in the 100bp flanking region.

[0023] 4.3 Screening results: Nine SNP sites that can be used for the identification of Guoqin No. 1 were finally obtained, as shown in Table 1.

[0024] Table 1. Marker information for 9 SNP sites used to identify Guoqin No. 1

[0025] It should be noted that in Table 1, Ref is the reference genome genotype; Alt is the validation result; PIC is the polymorphism information content; MAF is the minor allele frequency; and Pi is the nucleotide diversity.

[0026] In this embodiment of the invention, the specific information of the nucleotide sequences of the SNP sites corresponding to SNP1-SNP9 (SEQ ID NO.1~SEQ ID NO.9) is shown in Table 2, with the bases of the SNP sites in square brackets: Table 2

[0027] By comparing with the reference genome GWHDEDD00000000 (accessed by the National Center for Biotechnology Information, accession number GWHDEDD00000000), the genotype order of SNP loci corresponding to SNP1-SNP9 in the Guoqin No. 1 variety was determined to be: AA, TT, GG, CC, TT, TT, AA, GG, TT.

[0028] The genotypes of 9 SNP sites of the new Scutellaria baicalensis variety Guoqin No. 1 were converted into digital codes (using numbers 0-4 to encode the bases N, A, G, C, and T), where A=1, T=2, G=3, C=4, and N=0, resulting in the molecular ID number: 114422334444112244.

[0029] Example 2 The new Scutellaria baicalensis variety Guoqin No. 1 was identified using the SNP locus combinations of SNP1-SNP9 obtained from Example 1.

[0030] S1: Collect Scutellaria baicalensis seeds and seedlings sold on the market, and add the selected Guoqin No. 1 sample. The information of the collected Scutellaria baicalensis samples is shown in Table 3.

[0031] Table 3. Information on Scutellaria baicalensis seed and seedling test samples

[0032] S2: DNA was extracted from the samples using a universal genomic DNA extraction kit (magnetic bead method, China Migi-Yuhua kit) to obtain total DNA from the Scutellaria baicalensis samples. The extracted total DNA was detected by 1% agarose gel electrophoresis using a DYY-6C electrophoresis system, and the DNA concentration was detected by a Nanodrop UV spectrophotometer. The retention absorbance ratio (OD260 / 280) was between 1.8 and 2.2. Samples with a concentration greater than 50 ng / μL and a total amount greater than 2 μg were detected by Qubit 3.0.

[0033] S3: After the DNA sample passed the initial testing, it was randomly fragmented using a Covaris ultrasonic disruptor. The entire library preparation process then involved end repair, A-tailing, sequencing adapter addition, purification, and PCR amplification. After library construction, preliminary quantification was performed using Qubit 3.0 to dilute the library. Subsequently, the insert fragments were detected using an Agilent 2100. Once the insert fragment size met expectations, the effective concentration of the library was accurately quantified using Q-PCR to ensure library quality.

[0034] S4: After the libraries pass the test, different libraries are mixed according to the effective concentration and target data volume requirements. The libraries are then clustered using a cBOT clusterer and the Illumina NovaSeq high-throughput sequencing platform is used to perform high-throughput sequencing on the libraries to obtain raw sequencing data.

[0035] S5: The raw sequencing data (FASTQ format) is filtered using Trimmomatic to remove low-quality bases (HEADCROP: 10) and adapter contaminants, generating high-quality Clean Reads. Data quality is assessed using FastQC, including base quality distribution, GC content, and sequence reproducibility, ensuring that the Q30 base percentage is ≥90%, with no excessive shifts or contamination, yielding DNA quality control sequences. Specifically, the ILLUMINACLIP parameter can be used to remove street sequences, etc.

[0036] S6: Use BWA (v 0.7.13-r1126) software to align the quality control DNA sequence that has passed quality control to the Scutellaria baicalensis reference genome GWHDEDD00000000 with the default parameters. Based on the alignment results, use samtools software (version 0.1.18) to calculate the genotype of the target site. Finally, use Annovar software to annotate the mutation sites.

[0037] S7: Determine the genotype of the sample to be identified at the same SNP locus position based on the relevant location information of the SNP locus of Guoqin No. 1 in Table 1. If the genotype of the SNP locus corresponding to the sample to be identified is consistent with the genotype of the 9 SNP loci using SNP1-SNP9, then the sample to be identified is Guoqin No. 1.

[0038] S8: The obtained SNP sites are encoded according to the numbers 0-4 for the bases N, A, G, C, and T to obtain molecular identification. The specific sample SNP site genotype information and molecular identification are shown in Table 4.

[0039] Table 4. SNPs and coding information of Scutellaria baicalensis samples.

[0040] S9: The molecular identification codes of the samples to be identified were compared with those of Guoqin No. 1. The results showed that the Scutellaria baicalensis seeds and seedlings collected in the market did not match the molecular identification codes of Guoqin No. 1 and were not Guoqin No. 1 samples. The selected Guoqin No. 1 samples matched the molecular identification codes of Guoqin No. 1 and were Guoqin No. 1 samples. As can be seen from Table 4, the molecular identification codes of all selected Guoqin No. 1 samples in this embodiment were the same as those of Guoqin No. 1. There were significant differences in molecular identification codes between different varieties. This result is consistent with the actual situation, indicating that the accuracy of the identification results using the SNP locus combination of this invention is 100%.

[0041] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A combination of SNP molecular markers for identifying the new Scutellaria baicalensis variety, Guoqin No. 1, characterized in that, The SNP molecular marker combination consists of 9 SNP sites, SNP1-SNP9: SNP1 is located at physical position 3566028 on chromosome Sbai1, in gene Sbai1A35T79, and its polymorphism is T / A. SNP2 is located at physical position 1872824 on chromosome Sbai4, and the gene is Sbai4C18T15. Its polymorphism is A / T. SNP3 is located at physical position 2351968 on chromosome Sbai4, in gene Sbai4A23T90, and its polymorphism is A / G; SNP4 is located at physical position 8349245 on chromosome Sbai4, and the gene is Sbai4C83T14. Its polymorphism is C / T. SNP5 is located at physical position 9793077 on chromosome Sbai4, in gene Sbai4A98T73, and its polymorphism is C / T. SNP6 is located at physical position 18269593 on chromosome Sbai4, and the gene is Sbai4C182T8, with a polymorphism of C / T. SNP7 is located at physical position 22292483 on chromosome Sbai4, and the gene is Sbai4A223T75. Its polymorphism is G / A. SNP8 is located at physical position 25585245 on chromosome Sbai4, and the gene is Sbai4P255T65. Its polymorphism is G / C. SNP9 is located at physical position 37711547 on chromosome Sbai4, in gene Sbai4C377T4, and its polymorphism is C / T.

2. The SNP molecular marker combination according to claim 1, characterized in that, The nucleotide sequences of the nine SNP sites from SNP1 to SNP9 are shown in SEQ ID NO.1 to SEQ ID NO.9, respectively. The bases of the nine SNP sites from SNP1 to SNP9 correspond to the 101st position of the sequences shown in SEQ ID NO.1 to SEQ ID NO.

9.

3. A method for obtaining and identifying SNP molecular marker combinations for the new Scutellaria baicalensis variety, Guoqin No. 1, characterized in that, Includes the following steps: Obtain Scutellaria baicalensis samples, including Guoqin No. 1, and extract total DNA from each Scutellaria baicalensis sample; The extracted total DNA was subjected to quality testing. Sequencing libraries were constructed using DNA samples that met the quality standards, and the quality of the libraries was verified. The validated libraries were subjected to high-throughput sequencing and quality control processing to obtain DNA quality control sequences. The DNA quality control sequence was compared with the Scutellaria baicalensis reference genome GWHDEDD00000000 to determine the nine SNP molecular site marker combinations of SNP1-SNP9 as described in claim 1.

4. The method for obtaining and identifying the SNP molecular marker combination of the new Scutellaria baicalensis variety Guoqin No. 1 according to claim 3, characterized in that, A qualified DNA sample must meet at least the following conditions: The OD260 / 280 ratio for nucleic acid purity testing of DNA is between 1.8 and 2.

2. The nucleic acid concentration of the DNA is not less than 50 ng / μL, and the total content is not less than 2 μg.

5. The method for obtaining and identifying the SNP molecular marker combination of the new Scutellaria baicalensis variety Guoqin No. 1 according to claim 3, characterized in that, The quality control process includes: The high-throughput sequencing data were filtered to ensure that the proportion of Q30 bases was not less than 90%.

6. A method for constructing the molecular identity card of a new Scutellaria baicalensis variety, Guoqin No. 1, characterized in that, Includes the following steps: The nine SNP molecular site marker combinations of SNP1-SNP9 described in claim 1 are obtained by the method according to any one of claims 3-5; The genotypes of the nine SNP loci (SNP1-SNP9) were sequentially coded digitally to obtain the molecular identification of Guoqin No.

1.

7. The method for constructing the molecular identity card of the new Scutellaria baicalensis variety Guoqin No. 1 according to claim 6, characterized in that, The genotype order of the nine SNP loci from SNP1 to SNP9 is AA, TT, GG, CC, TT, TT, AA, GG, TT.

8. The method for constructing the molecular identity card of the new Scutellaria baicalensis variety Guoqin No. 1 according to claim 6, characterized in that, The genotypes of the nine SNP loci (SNP1-SNP9) in Guoqin No. 1 were sequentially digitally encoded, including: The bases of the nine SNP sites (SNP1-SNP9) in Guoqin No. 1 were digitally encoded using the numbers 0-4, where A=1, T=2, G=3, C=4, and N=0. The molecular identity card of Guoqin No. 1 is generated by sequentially cascading the digital codes of the nine SNP sites from SNP1 to SNP9.

9. A molecular identification card for a new Scutellaria baicalensis variety, Guoqin No. 1, characterized in that, The molecular identity card is constructed by the method described in any one of claims 6-8.

10. A method for identifying a new Scutellaria baicalensis variety, Guoqin No. 1, characterized in that, The method includes the following steps: Obtain the genotype of the sample to be identified. If the genotype of the sample to be identified is consistent with the genotype of the nine SNP loci of SNP1-SNP9 in claim 7 or 8, it is determined to be the new Scutellaria baicalensis variety Guoqin No.

1. Alternatively, the method may include the following steps: Obtain the molecular identification of the sample to be identified. If the molecular identification of the sample to be identified is consistent with the molecular identification in claim 7 or 8, it is determined to be the new Scutellaria baicalensis variety Guoqin No. 1.

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