Method for constructing SNP molecular marker combination and molecular identity card of new scutellaria baicalensis george variety guoqin no.1 and application thereof

Abstract

The present application relates to the field of molecular biology technology, in particular to the construction method and application of SNP molecular marker combination and molecular identity card for identifying new variety of Scutellaria baicalensis Georgi Guo Qin No.1. The present application determines the SNP molecular marker combination for identifying new variety of Scutellaria baicalensis Georgi Guo Qin No.1 by using SNP molecular marker technology, and constructs the molecular identity card of Guo Qin No.1, which widens the application of molecular identity card in the identification of Chinese herbal medicine varieties, lays a foundation for the establishment of molecular identity card in the identification of other Chinese herbal medicine varieties and the establishment of variety source tracing system, and provides theoretical support for the establishment of variety identification standard.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology technology, and particularly relates to a SNP molecular marker combination for identifying a new Scutellaria baicalensis Georgi variety Guoqin No. 1 and a construction method and application of a molecular identity card. BACKGROUND

[0002] As a bulk medicinal material, Scutellaria baicalensis Georgi has the effects of clearing heat and drying dampness, purging fire and detoxifying, and stopping bleeding. In December 2022, Guoqin No. 1 was awarded the plant new variety right by the State Forestry and Grassland Administration, which is the first Scutellaria baicalensis Georgi plant new variety awarded the variety right in China, but there is still a lack of identification method for the variety. Although traditional traits and physiological and biochemical indicators can identify medicinal material germplasm resources to some extent, it is difficult for traditional methods and physical and chemical methods to accurately identify the same variety from different sources and similar varieties and pseudo varieties with close genetic relationship. With the deepening of the breeding of new varieties of medicinal materials, the accurate identification of new varieties of medicinal materials and mainstream homegrown varieties and wild resources is more conducive to the healthy development of the medicinal material industry. SUMMARY

[0003] In view of the above problems, in a first aspect, the application discloses a SNP molecular marker combination for identifying a new Scutellaria baicalensis Georgi variety Guoqin No. 1, which is composed of nine SNP sites of SNP1-SNP9:

[0004] SNP1 is located at a physical position of 3566028 of chromosome Sbai1, the gene is Sbai1A35T79, and the polymorphism is T / A;

[0005] SNP2 is located at a physical position of 1872824 of chromosome Sbai4, the gene is Sbai4C18T15, and the polymorphism is A / T;

[0006] SNP3 is located at a physical position of 2351968 of chromosome Sbai4, the gene is Sbai4A23T90, and the polymorphism is A / G;

[0007] SNP4 is located at a physical position of 8349245 of chromosome Sbai4, the gene is Sbai4C83T14, and the polymorphism is C / T;

[0008] SNP5 is located at a physical position of 9793077 of chromosome Sbai4, the gene is Sbai4A98T73, and the polymorphism is C / T;

[0009] SNP6 is located at a physical position of 18269593 of chromosome Sbai4, the gene is Sbai4C182T8, and the polymorphism is C / T;

[0010] SNP7 is located at the physical position of 22292483 of chromosome Sbai4, the gene is Sbai4A223T75, and the polymorphism is G / A;

[0011] SNP8 is located at the physical position of 25585245 of chromosome Sbai4, the gene is Sbai4P255T65, and the polymorphism is G / C;

[0012] SNP9 is located at the physical position of 37711547 of chromosome Sbai4, the gene is Sbai4C377T4, and the polymorphism is C / T.

[0013] Further, the nucleotide sequences at the 9 SNP sites of SNP1-SNP9 are shown in SEQ ID NO. 1~SEQ ID NO. 9 respectively.

[0014] The base of the 9 SNP sites of SNP1-SNP9 corresponds to the 101st position of the sequence shown in SEQ ID NO. 1~SEQ ID NO. 9.

[0015] In a second aspect, the present application provides a method for obtaining a SNP molecular marker combination of the new variety of Scutellaria baicalensis Georgi Guoqin No. 1, comprising the following steps:

[0016] Obtain Scutellaria baicalensis Georgi samples including Guoqin No. 1, and extract total DNA of each Scutellaria baicalensis Georgi sample;

[0017] Quality detection is performed on the extracted total DNA, and a sequencing library is constructed with the DNA sample of qualified quality, and the quality of the library is verified;

[0018] High-throughput sequencing is performed on the verified library, and quality control processing is performed to obtain a DNA quality control sequence;

[0019] The DNA quality control sequence is compared with the Scutellaria baicalensis Georgi reference genome GWHDEDD00000000 to determine the 9 SNP molecular site marker combination of SNP1-SNP9.

[0020] Further, the DNA sample of qualified quality at least meets the following conditions:

[0021] The OD260 / 280 ratio of the nucleic acid purity detection of the DNA is between 1.8-2.2;

[0022] The nucleic acid concentration of the DNA is not less than 50 ng / μL, and the total content is not less than 2 μg.

[0023] Further, the quality control processing comprises:

[0024] Filtering processing is performed on the data after high-throughput sequencing, and the proportion of Q30 bases is not less than 90%.

[0025] In a third aspect, the present application provides a method for constructing a molecular identity card of Scutellaria baicalensis Georgi new variety Guoqin No.1, comprising the following steps:

[0026] According to the method for obtaining the SNP molecular marker combination for identifying the Scutellaria baicalensis Georgi new variety Guoqin No.1, the 9 SNP molecular site marker combinations of SNP1-SNP9 are obtained.

[0027] The genotypes of the 9 SNP sites of SNP1-SNP9 are sequentially digitally coded to obtain the molecular identity card of Guoqin No.1.

[0028] Further, the genotype sequence of the 9 SNP sites of SNP1-SNP9 is AA, TT, GG, CC, TT, TT, AA, GG, and TT.

[0029] Further, the sequential digital coding of the genotypes of the 9 SNP sites of SNP1-SNP9 in Guoqin No.1 comprises:

[0030] The bases of the 9 SNP sites of SNP1-SNP9 in Guoqin No.1 are sequentially digitally coded by using numbers 0-4, wherein A=1, T=2, G=3, C=4, and N=0.

[0031] The digital codes of the 9 SNP sites of SNP1-SNP9 are sequentially connected to generate the molecular identity card of Guoqin No.1.

[0032] In a fourth aspect, the present application provides a molecular identity card of Scutellaria baicalensis Georgi new variety Guoqin No.1, which is constructed by the method for constructing a molecular identity card of Scutellaria baicalensis Georgi new variety Guoqin No.1.

[0033] In a fifth aspect, the present application provides a method for identifying Scutellaria baicalensis Georgi new variety Guoqin No.1, comprising the following steps:

[0034] The genotype of the sample to be identified is obtained, and if the genotype of the sample to be identified is consistent with the genotype of the 9 SNP sites of SNP1-SNP9, it is determined as Scutellaria baicalensis Georgi new variety Guoqin No.1.

[0035] Alternatively, the method comprises the following steps:

[0036] The molecular identity card of the sample to be identified is obtained, and if the molecular identity card of the sample to be identified is consistent with the molecular identity card of Guoqin No.1, it is determined as Scutellaria baicalensis Georgi new variety Guoqin No.1.

[0037] The present application has the following beneficial effects:

[0038] The application utilizes SNP molecular marker technology to construct a molecular identity card of a new variety of Chinese herbal medicine Scutellaria baicalensis Georgi, Guo Qin No. 1, which can quickly and accurately identify the authenticity of the parent and its offspring seeds of the new variety of Scutellaria baicalensis Georgi, Guo Qin No. 1, protect the plant new variety right, prevent fake and inferior variety seeds from entering the market, and ensure the safety and reliability of excellent seeds. Meanwhile, the application of the molecular identity card in the variety identification of Chinese herbal medicines is widened, which lays a foundation for the establishment of the molecular identity card in the variety identification and seed source tracing system of other Chinese herbal medicines, provides theoretical support for the establishment of the variety identification standard, and ensures the safety and reliability of the introduction of Guo Qin No. 1, thereby providing an important guarantee for the high-quality development of Scutellaria baicalensis Georgi industry.

[0039] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the present application. The objects and other advantages of the present application will be realized and attained by the structure particularly pointed out in the written description and claims. DETAILED DESCRIPTION

[0040] In order to make the objectives, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are some embodiments of the present application, but not all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0041] Embodiment 1

[0042] Screening of SNP site combination of new variety of Scutellaria baicalensis Georgi, Guo Qin No. 1:

[0043] Step 1: Collect 107 tissue sample germplasms of Scutellaria baicalensis Georgi including Guo Qin No. 1;

[0044] Step 2: Total DNA extraction, quality detection and genotyping;

[0045] 2.1 Universal genomic DNA extraction kit (magnetic bead method, China Meiji Huaxia Reagent Kit) is used for sample DNA extraction to obtain total DNA of Scutellaria baicalensis Georgi samples;

[0046] 2.2 DYY-6C electrophoresis instrument is used for 1% agarose gel electrophoresis to detect the total DNA of Scutellaria baicalensis Georgi samples;

[0047] 2.3 Nanodrop ultraviolet spectrophotometer is used to detect the concentration of total DNA of Scutellaria baicalensis Georgi samples, the light absorption ratio (OD260 / 280) is between 1.8 and 2.2, the Qubit 3.0 detects the concentration greater than 50 ng / μL, and the total amount is greater than 2 ug.

[0048] 2.4 After the DNA sample is qualified, use the Covaris ultrasonic crusher to randomly break it, and then go through the steps of end repair, A tail addition, sequencing adapter addition, purification, PCR amplification, etc. to complete the entire library preparation work. After the library construction is completed, use Qubit 3.0 to perform preliminary quantification, dilute the library, and then use Agilent 2100 to detect the insert size of the library. After the insert size meets the expected value, use Q-PCR method to accurately quantify the effective concentration of the library to ensure the quality of the library.

[0049] 2.5 After the library is qualified, according to the effective concentration and the target data volume required, pool different libraries to the Flow cell, and after cBOT clustering, use Illumina high-throughput sequencing platform Illumina NovaSeq X plus for sequencing.

[0050] Step 3: Quality control of sequencing data and sequence alignment;

[0051] 3.1 The original sequencing data (FASTQ format) is filtered by Trimmomatic to remove low-quality bases (HEADCROP: 10) and remove adapter contamination to generate high-quality Clean Reads. The data quality is evaluated by FastQC, including base quality distribution, GC content, sequence repeatability, etc. to ensure that the Q30 base ratio is ≥90%, and there is no excessive deviation or contamination.

[0052] 3.2 Use BWA (v0.7.13-r1126) software to align the qualified sequences to the reference genome GWHDEDD00000000 (included in the National Bioinformation Center, accession number GWHDEDD00000000) with default parameters. According to the alignment results, use samtools software (version 0.1.18) to calculate the genotype results of the target site, and finally use Annovar software for gene annotation of mutation sites.

[0053] Step 4: SNP site screening;

[0054] 4.1 A total of 1,121,566 original SNP sites were obtained through original data analysis. In order to ensure the accuracy and reliability of the subsequent analysis results, strict quality filtering, deletion filtering, minor allele frequency filtering were performed on all SNP sites, and di-allele and 4DTv sites were also screened. After multiple rounds of strict screening, 24,783 high-quality SNP sites were finally left.

[0055] 4.2 Filter and screen the candidate SNPs from the high-quality SNP sites by using the PLINK software. The specific criteria are as follows: a. biallelic sites; b. 4-fold degenerate SNPs; c. heterozygosity <0.2; d. missing rate <0.2; e. minor allele frequency (MAF) >0.05; f. no InDel or SNP in the 100 bp flanking region.

[0056] 4.3 Screening results: finally, 9 SNP sites that can be used for identification of Guoqing No. 1 are obtained, as shown in Table 1.

[0057] Table 1 9 SNP site marker information for identifying Guoqing No. 1

[0058]

[0059] It should be noted that in Table 1, Ref is the reference genome genotype; Alt is the verification result; PIC is the polymorphic information content; MAF is the minor allele frequency; and Pi is the nucleotide diversity.

[0060] In the embodiment of the present application, the specific information of the nucleotide sequences of the SNP sites corresponding to SNP1-SNP9 of SEQ ID NO. 1-SEQ ID NO. 9 is shown in Table 2, and the bases in the square brackets are the SNP sites:

[0061] Table 2

[0062]

[0063] By comparing with the reference genome GWHDEDD00000000 (included in the National Bioinformatics Center, accession number GWHDEDD00000000), the genotype order of the SNP sites corresponding to SNP1-SNP9 of the Guoqing No. 1 variety is determined as follows: AA, TT, GG, CC, TT, TT, AA, GG, and TT.

[0064] The genotypes of the 9 SNP sites of the new Scutellaria baicalensis Georgi variety Guoqing No. 1 are converted into digital codes (using the numbers 0-4 to encode the bases N, A, G, C, and T), wherein A=1, T=2, G=3, C=4, and N=0, and the molecular identity card is obtained as: 114422334444112244.

[0065] Example 2

[0066] The SNP1-SNP9 SNP site combination obtained by screening in Example 1 is used to identify the new Scutellaria baicalensis Georgi variety Guoqing No. 1.

[0067] S1: Collect the market selling Scutellaria baicalensis Georgi seed seedlings, and add the selected Guo Qin No. 1 sample. The collected Scutellaria baicalensis Georgi sample information is shown in Table 3.

[0068] Table 3 Scutellaria baicalensis Georgi seed seedling detection sample information

[0069]

[0070] S2: The total DNA of the Scutellaria baicalensis Georgi sample was obtained by using a general genomic DNA extraction kit (magnetic bead method, China Meiji Huaxia Reagent Kit) for sample DNA extraction. The total DNA after extraction was detected by using a DYY-6C electrophoresis instrument, using 1% agarose gel electrophoresis, and the concentration of the DNA was detected by using a Nanodrop ultraviolet spectrophotometer. The light absorption ratio (OD260 / 280) was between 1.8 and 2.2, the concentration was greater than 50 ng / μL, and the total amount of the DNA sample was greater than 2 ug.

[0071] S3: After the DNA sample detection is qualified, the Covaris ultrasonic crusher is used for random breaking, and then the end repair, A tail adding, sequencing adapter adding, purification, PCR amplification and other steps are used to complete the whole library preparation work. After the library construction is completed, the Qubit3.0 is used for preliminary quantification, the library is diluted, and then the Agilent 2100 is used for detecting the insert fragment of the library. After the insert fragment size meets the expectation, the Q-PCR method is used for accurate quantification of the effective concentration of the library to ensure the quality of the library.

[0072] S4: After the library detection is qualified, different libraries are mixed according to the effective concentration and the target data amount required for the next machine, and then the cBOT is used for clustering the library, and the Illumina high-throughput sequencing platform Illumina NovaSeq is used for high-throughput sequencing of the library to obtain the original sequencing data.

[0073] S5: The original sequencing data (FASTQ format) is filtered by Trimmomatic, low-quality bases (HEADCROP: 10) are cut off, and adapter contamination is removed to generate high-quality Clean Reads. The data quality is evaluated by FastQC, including base quality distribution, GC content, sequence repeatability and other indicators to ensure that the Q30 base ratio is ≥90%, there is no excessive deviation or contamination, and the DNA quality control sequence is obtained. Specifically, the ILLUMINACLIP parameter can be used to remove street sequences and the like.

[0074] S6: The qualified DNA quality control sequence is aligned to the Huangqi reference genome GWHDEDD00000000 using BWA (v0.7.13-r1126) software, and the parameters are default parameters. According to the alignment results, the genotype results of the target site are calculated by samtools software (version 0.1.18), and finally the mutation site is annotated by Annovar software.

[0075] S7: The genotype of the sample to be identified at the same SNP site position is determined according to the position information of the SNP site of Guoqing No. 1 in Table 1. If the genotype of the SNP site corresponding to the sample to be identified is consistent with the genotype of the nine SNP sites of SNP1-SNP9, it is determined that the sample to be identified is Guoqing No. 1.

[0076] S8: The obtained SNP site is encoded according to the bases N, A, G, C and T, and the molecular identity card is obtained. The SNP site genotype information and the molecular identity card of the specific sample are shown in Table 4.

[0077] Table 4 SNP and encoding information of Huangqi detection sample

[0078]

[0079] S9: The molecular identity card of the sample to be identified is compared with the molecular identity card of Guoqing No. 1. The results show that the Huangqi seed seedlings collected on the market do not match the molecular identity card of Guoqing No. 1, and do not belong to Guoqing No. 1 sample; the selected Guoqing No. 1 sample matches the molecular identity card of Guoqing No. 1, and belongs to Guoqing No. 1 sample. As can be seen from Table 4, the molecular identity cards of all the selected Guoqing No. 1 samples in this embodiment are the same as the molecular identity card of Guoqing No. 1, and the molecular identity cards of different varieties are quite different. The results are consistent with the actual situation, and the accuracy rate of the identification results of the SNP site combination of the present application is 100%.

[0080] Although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. 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 combination of 9 SNP molecular site markers from SNP1 to SNP9. 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 position 101 of the sequences shown in SEQ ID NO.1 to SEQ ID NO.

9. 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 method for obtaining and identifying the SNP molecular marker combination of the new Scutellaria baicalensis variety Guoqin No. 1 according to claim 1, 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.

3. The method for obtaining and identifying the SNP molecular marker combination of the new Scutellaria baicalensis variety Guoqin No. 1 according to claim 1, 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%.

4. 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 are obtained according to the method of any one of claims 1-3; The genotypes of the nine SNP loci (SNP1-SNP9) were sequentially coded digitally to obtain the molecular identification of Guoqin No.

1.

5. The method for constructing the molecular identity card of the new Scutellaria baicalensis variety Guoqin No. 1 according to claim 4, 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.

6. The method for constructing the molecular identity card of the new Scutellaria baicalensis variety Guoqin No. 1 according to claim 4, 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.

7. 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 (SNP1-SNP9) in claim 1, 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 card of the sample to be identified. If the molecular identification card of the sample to be identified is consistent with the molecular identification card constructed according to the method of any one of claims 4-6, it is determined to be the new Scutellaria baicalensis variety Guoqin No. 1.

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