CN112322779B - SNP molecular marker for identifying liquorice produced in Xinjiang, and method and application thereof - Google Patents

Abstract

The invention relates to an SNP molecular marker for identifying liquorice produced in Xinjiang, and a method and application thereof, wherein the molecular marker comprises 40 SNP molecular markers, and the nucleotide sequences of the 40 SNP molecular markers are shown in SEQ ID NO.1-SEQ ID NO. 40. The SNP molecular marker for identifying liquorice produced in Xinjiang and the method and application thereof adopt the genomics technology to obtain the SNP marker on the liquorice genome level, and can quickly and accurately determine whether the detected liquorice producing area is the Xinjiang producing area.

Description

SNP molecular marker for identifying liquorice produced in Xinjiang, and method and application thereof

Technical Field

The invention belongs to the field of molecular biological molecular markers, and particularly relates to an SNP molecular marker for identifying liquorice produced in Xinjiang, and a method and application thereof.

Background

The liquorice is a traditional Chinese medicine commonly used in bulk, is sweet and mild in taste, and has the effects of tonifying spleen and qi, clearing heat and removing toxicity, eliminating phlegm and stopping cough, relieving spasm and pain and harmonizing the medicines. Mainly treats the symptoms of weakness of the spleen and the stomach, lassitude and hypodynamia, palpitation and shortness of breath, cough and excessive phlegm, abdominal and limb spasm and pain, carbuncle, swelling, sore and the like, and relieves the toxicity and the intensity of the medicaments. The liquorice has the nineteen-ingredient nine-grass, so the liquorice appears most frequently in various compound Chinese medicaments and mainly has the effect of harmonizing the medicaments. The major producing areas of licorice include inner Mongolia, Xinjiang, Gansu and Ningxia, and the northeast area has a small amount of distribution. The quality and the efficacy of the liquorice medicinal materials in different producing areas are different, and the price of the liquorice seeds, the liquorice medicinal materials and the like in different producing areas are obviously different, and particularly, the price of the liquorice seeds and the liquorice medicinal materials produced in Xinjiang is lower than that of the liquorice seeds and the liquorice medicinal materials produced in other producing areas.

The identification of the production place of the liquorice is always a technical difficulty for identifying the liquorice species and is also an industrial problem faced by the traditional Chinese medicine resource industry at present. At present, the method of identifying characteristics and microscopic identification is mostly used for identifying the production place of liquorice, and the method depends on personal experience and subjective judgment, so that the accuracy is not high, and the technology is not strong in popularization.

However, in Xinjiang area of the main production area of liquorice, how to distinguish the liquorice produced by the Xinjiang area from the liquorice produced by other areas is a problem which needs to be solved urgently by traditional Chinese medicine identification personnel.

Disclosure of Invention

Aiming at the problems, the invention provides an SNP molecular marker for identifying liquorice produced in Xinjiang, and a method and application thereof.

An SNP molecular marker for identifying liquorice produced in Xinjiang comprises 40 SNP molecular markers, and the nucleotide sequences of the 40 SNP molecular markers are shown in SEQ ID No.1-SEQ ID No. 40.

Further, the bases of the 40 SNP molecular markers are shown below.

A method for identifying liquorice producing area by using SNP molecular markers for identifying liquorice produced in Xinjiang, which comprises the following steps:

obtaining DNA of a sample to be detected;

carrying out library building and sequencing on the qualified sample DNA to obtain sample DNA sequencing data;

performing quality control on the sample DNA sequencing data to obtain sample DNA quality control data;

comparing the sample DNA quality control data sample with a reference sequence, removing the duplication and carrying out SNP detection to obtain a SNP gene of a sample to be detected;

comparing the SNP genes of the sample to be detected at the same chromosome position with the SNP molecular marker of liquorice produced in Xinjiang, judging whether 19-20 SNP genes of the sample to be detected are the same as the SNP molecular markers of the liquorice produced in Xinjiang, SEQ ID NO.1-SEQ ID NO.20, and simultaneously judging whether 19-20 SNP genes of the sample to be detected are different from the SNP molecular markers of the liquorice produced in Xinjiang, SEQ ID NO.21-SEQ ID NO. 40:

if yes, judging that the sample to be detected is the liquorice in the Xinjiang producing area; and if not, the sample to be detected is the liquorice in the non-Xinjiang producing area.

Further, the sequencing of the sample DNA employs high-throughput sequencing.

Further, the quality control of the sample DNA sequencing data comprises:

filtering the original reads of the sample DNA sequencing data;

the filtering rules include:

removing the linker sequence;

removing reads with the number of N in the sequence being less than or equal to 2% of the length of the sequence;

reads with sequencing quality below 20 and more than 40% of the sequence length are removed.

Further, after comparing the sample DNA quality control data sequence with the reference sequence and removing duplication, performing SNP detection to obtain the SNP gene of the sample to be detected comprises:

taking a genome of liquorice in a public database as a reference sequence, and performing sequence comparison by using comparison software to obtain a bam file;

sorting the bam files by using sorting software, and removing a repeated sequence;

processing the sequenced and de-duplicated bam files by using genotyping software to obtain the preliminary SNP genotype of the sample to be detected;

and reducing the error rate of the preliminary SNP genotype of the sample by using the parameters of QUAL >100 & & FS <10 & & MQ >40 to obtain the SNP genotype of the sample to be detected.

The invention also comprises application of the SNP molecular marker in identifying liquorice produced in Xinjiang.

The invention also comprises a gene chip or a kit containing the SNP molecular marker.

The SNP molecular marker for identifying liquorice produced in Xinjiang and the method and application thereof adopt the genomics technology to obtain the SNP marker on the liquorice genome level, and can quickly and accurately determine whether the detected liquorice producing area is the Xinjiang producing area. Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof.

Detailed Description

In order to make the objects, 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 with reference to the embodiments of the present invention, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Obtaining liquorice SNP molecular markers:

DNA extraction

In this experiment, the DNA extraction was performed on the licorice leaf material by the mCTAB method (modified CTAB method). The specific extraction steps are as follows:

(1) putting 100 mg of fresh plant leaves of liquorice into a 2.0 mL centrifuge tube, adding a small amount of quartz sand and a magnetic bead, immersing the fresh leaves into liquid nitrogen, freezing for about 1 min, and putting the leaves into a grinder to be ground into fine powder for later use;

(2) adding 1.5mL buffer A (0.1M Tris-HCl pH 8.0; 5mM EDTA; 0.25M NaCl; 1% PVP-40) solution into the ground material, repeatedly inverting the centrifuge tube to homogenize and mix the solution and the material powder, performing ice bath for 10min, and then homogenizing and mixing the precipitated powder for 3-10 times again. Centrifuging at 10000 Xg, and removing supernatant;

(3) repeating the step (2) once, adding 800 μ L of preheated 2% CTAB (0.1M Tris-HCl pH8.0; 1.4M NaCl; 25mM EDTA; 2% (w/v) CTAB; 0.2% (v/v) beta-mercaptoethanol; 1% PVP-40) solution into the precipitate, placing the precipitate after being homogenized and suspended in the solution in a 65 ℃ water bath kettle for storing for 1.5-2h, and reversing the homogenized solution for 2-8 times;

(4) after centrifugation at 10000 Xg at room temperature, the supernatant was carefully poured into a new 2.0 mL centrifuge tube, an equal volume of CI (chloroform: isoamyl alcohol =24:1 (v/v)) solution was added and mixed on an inverted shaker for 10 min;

(5) after centrifugation at 10000 Xg, carefully absorbing the supernatant aqueous phase clear liquid into a new 2.0 mL centrifuge tube by using a pipette gun, adding an equal volume of CI solution, and mixing for 10min on an inverted shaker;

(6) after 10000 Xg centrifugation, carefully absorbing the supernatant aqueous phase clear liquid by a pipette gun to a new 1.5mL centrifuge tube, adding 0.6 volume times of ice-cold isopropanol, reversing the mixture, and storing in a refrigerator at-20 ℃ for more than 1 hour;

(7) taking out the centrifuge tube in the refrigerator, centrifuging at 10000 Xg, discarding supernatant, pouring on dry paper to control liquid drop as much as possible, adding 100 μ L RNase solution (100 mg/L), and storing at 37 deg.C for 0.5 h;

(8) sequentially adding 150 mu L of ddH2O, 50 mu L of 5M NaCl and 700 mu L of absolute ethyl alcohol into a centrifuge tube, fully mixing, centrifuging at 10000 Xg, discarding supernatant, and pouring on paper to dry;

(9) adding 600 μ L70% ethanol, mixing, centrifuging, removing supernatant, and repeating;

(10) ethanol was dried under vacuum, 100. mu.L TE was added to dissolve DNA, and the sample DNA concentration was diluted to 50 ng/. mu.L according to the Nanodrop assay.

2. Library construction sequencing

And randomly breaking the qualified DNA sample into 350bp fragments, and establishing a library by adopting a Huada self-research kit. After the library is qualified, BGI-SEQ high-throughput sequencing is carried out according to the effective concentration and the actual output demand of the library, and the original data of DNA sequencing is obtained. The DNA high-throughput sequencing can adopt any high-throughput sequencing platform, besides the BGI-SEQ platform, Illumina, SOLID, Pacbio, ONT (Oxford Nanopore technologies) platform and the like can be adopted.

3. Sequencing data quality control

The original reads obtained by sequencing contain linker sequences, low quality and reads containing N. To ensure the quality of the information analysis, the original reads must be filtered. The filtering rules include:

A. removing the linker sequence;

B. removing reads with the number of N in the sequence being less than or equal to 2% of the length of the sequence;

C. reads with a number of bases with a sequencing quality (MQ) below 20 exceeding 40% of the sequence length are removed.

4. Sequence alignment

The genome of liquorice in an NCBI public database is used as a reference sequence, BWA software is used for sequence alignment, and the alignment parameter is mem-t 8-M-R. The bam files obtained from the alignment were sorted using Samtools software and the duplicate sequences were removed.

SNP detection

After the bam file is obtained, the SNP genotype of the individual is obtained by utilizing the GATK software. To reduce the error rate of SNP detection, SNPs were screened "QUAL >100 & & FS <10 & & MQ > 40" using the following parameters. After screening and analysis, 40 SNP molecular markers associated with the glycyrrhiza uralensis of Xinjiang area are obtained, as shown in Table 1.

TABLE 1

6. Screening comparison

And detecting the genotypes of the 40 SNP sites of each sample to be detected. If all samples to be detected meet the following 95% detection standard of 40 sites, the samples to be detected are liquorice in Xinjiang district: namely, the same as any 19-20 of 1-20 SNP loci in table 1, and different from any 19-20 of 21-40 SNP loci in table 21, namely, the Xinjiang section liquorice.

Example 1

Sample information: liquorice in areas (NMGC 101, NMGC102, NMG201 and NMGC 202), Xinjiang (XJGC 101, XJGC102, XJGC201, XJGC202, XJGC301, XJGC302, XJGC303, XJGC401, XJGC402 and XJGC 403) and Gansu (GSGC 101, GSGC102, GSGC201 and GSGC 202) are collected respectively. The sample details are shown in Table 2.

And (3) identification:

1. sample DNA extraction

DNA extraction was performed on each sample licorice leaf material by the mCTAB method (modified CTAB method). The specific extraction steps are as follows:

(1) putting 100 mg of fresh plant leaves into a 2.0 mL centrifuge tube, adding a small amount of quartz sand and a magnetic bead, immersing the fresh leaves into liquid nitrogen, freezing for about 1 min, and grinding the fresh leaves into fine powder in a grinder for later use;

(2) 1.5mL of buffer A (0.1M Tris-HCl pH 8.0; 5mM EDTA; 0.25M NaCl; 1% PVP-40) solution was added to the ground material, the tube was inverted repeatedly to homogenize the solution and the material powder, and the mixture was ice-cooled for 10min, during which the precipitated powder was again homogenized several times. Centrifuging at 10000 Xg, and removing supernatant;

(3) repeating the step (2) once, adding 800 μ L of preheated 2% CTAB (0.1M Tris-HCl pH 8.0; 1.4M NaCl; 25mM EDTA; 2% (w/v) CTAB; 0.2%, (v/v) beta-mercaptoethanol; 1% PVP-40) solution into the precipitate, placing the precipitate after being homogenously suspended in the solution in a 65 ℃ water bath kettle for storing for 1.5-2h, and reversing the homogenated solution for several times;

(4) after centrifugation at 10000 Xg at room temperature, the supernatant was carefully poured into a new 2.0 mL centrifuge tube, an equal volume of CI (chloroform: isoamyl alcohol =24:1 (v/v)) solution was added and mixed on an inverted shaker for 10 min;

(5) after centrifugation at 10000 Xg, carefully absorbing the supernatant aqueous phase clear liquid into a new 2.0 mL centrifuge tube by using a pipette gun, adding an equal volume of CI solution, and mixing for 10min on an inverted shaker;

(6) after 10000 Xg centrifugation, carefully absorbing the supernatant aqueous phase clear liquid by a pipette gun to a new 1.5mL centrifuge tube, adding 0.6 volume times of ice-cold isopropanol, reversing the mixture, and storing in a refrigerator at-20 ℃ for more than 1 hour;

(7) taking out the centrifuge tube in the refrigerator, centrifuging at 10000 Xg, discarding supernatant, pouring on dry paper to control liquid drop as much as possible, adding 100 μ L RNase solution (100 mg/L), and storing at 37 deg.C for 0.5 h;

(8) sequentially adding 150 mu L of ddH2O, 50 mu L of 5M NaCl and 700 mu L of absolute ethyl alcohol into a centrifuge tube, fully mixing, centrifuging at 10000 Xg, discarding supernatant, and pouring on paper to dry;

(9) adding 600 μ L70% ethanol, mixing, centrifuging, removing supernatant, and repeating;

(10) ethanol was dried under vacuum, 100. mu.L TE was added to dissolve DNA, and the sample DNA concentration was diluted to 50 ng/. mu.L according to the Nanodrop assay.

2. Sample library construction sequencing

And (3) randomly breaking the qualified DNA sample into 350bp fragments, and constructing a library by using a kit. After the library is qualified, BGI-SEQ high-throughput sequencing is carried out according to the effective concentration and the actual output demand of the library, and the original data of DNA sequencing is obtained. The DNA high-throughput sequencing can adopt any high-throughput sequencing platform, besides the BGI-SEQ platform, Illumina, SOLID, Pacbio, ONT (Oxford Nanopore technologies) platform and the like can be adopted.

3. Sequencing data quality control

The original reads obtained by sequencing contain linker sequences, low quality and reads containing N. To ensure the quality of the information analysis, the original reads must be filtered. The filtering rules include:

A. removing the linker sequence;

B. removing reads with the number of N in the sequence being less than or equal to 2% of the length of the sequence;

C. removing reads with a number of bases with a sequencing quality (MQ) below 20 exceeding 40% of the sequence length;

4. and (5) aligning sample sequences.

And taking the genome of the liquorice in the NCBI public database as a reference sequence, and performing sequence comparison by using BWA software, wherein the comparison parameter is mem-t 8-M-R. The bam files obtained from the alignment were sorted using Samtools software and the duplicate sequences were removed.

And 5, SNP detection.

After the bam file is obtained, the SNP genotype of the individual is obtained by utilizing the GATK software. To reduce the error rate of SNP detection, we screened SNPs with the following parameters "QUAL >100 & & FS <10 & & MQ > 40".

6. Screening and comparing to obtain the liquorice produced in Xinjiang

Comparing the genome SNP information of the liquorice in the Xinjiang area and the liquorice in the Gansu area in the sample and the genome SNP information of the liquorice in the inner Mongolia area with the SNP information in the table 1, and finding that 19-20 of the first 20 SNP sites of the liquorice sample in the Xinjiang area are the same as the 1 st-20 th site in the table 1, and 19-20 of the last 20 SNP sites are different from the 21 st-40 th site in the table 1.

Therefore, compared with the SNP information in the table 1, if the nucleotide at the same position of the sample to be detected does not meet any one judgment standard, namely the first 20 position point information of the sample does not meet the requirement that 19-20 position points are the same as the 1 st-20 position points in the table 1, or the last 20 position point information does not meet the requirement that 19-20 position points are different from the 21 st-40 position points in the table 1, the licorice group to be detected is the non-Xinjiang section licorice.

Example 2

Sample information: collecting liquorice in inner Mongolia (NMGC 103, NMGC 203), Xinjiang (XJGC 103, XJGC 203) and Gansu (GSGC 103, GSGC 203) areas respectively. The sample details are shown in Table 2.

TABLE 2 sample specific information

Identifying glycyrrhiza uralensis in Xinjiang district and glycyrrhiza uralensis in other districts by the SNP sites shown in Table 1 in the following Table 1, and obtaining SNP site information of each sample at the same position with reference to the SNP site information obtaining method in example 1.

The SNP genes at the same position of each sample were compared with the SNP gene of Xinjiang licorice in Table 1, and the comparison results are shown in Table 3.

TABLE 3 SNP genotyping results

Note: a single A or C or G or T represents a homozygote.

The identification results in Table 3 show that the XJGC103 and XJGC203 genes of the sample are completely the same as the Xinjiang licorice SNP gene 1-20 in Table 1 and are completely different from the Xinjiang licorice SNP gene 21-40, and other samples cannot simultaneously meet the condition that two judgments are available, which indicates that the sample XJGC103, the XJGC203 is the Xinjiang producing area, and other samples are non-Xinjiang producing areas.

Through 40 SNP genes in table 1 and a method for identifying the producing area of Xinjiang liquorice by applying the genes, the 40 SNP genes are compared with SNP genes at the same position of the liquorice to be detected, and the condition that 19-20 of the first 20 SNP sites are the same as the 1 st-20 th site in table 1 and 19-20 of the last 20 SNP sites are different from the 21 st-40 th site in table 1 is determined to be the Xinjiang liquorice in the area of Xinjiang liquorice. The method for identifying the liquorice produced in Xinjiang is simple and quick, has accurate result, and can effectively identify the liquorice in non-Xinjiang producing areas.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

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CHINA TRADITIONAL CHINESE MEDICINE SEED & SEEDLING Co.,Ltd.

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Claims (7)

1. The SNP molecular marker for identifying liquorice produced in Xinjiang is characterized by comprising 40 SNP molecular markers, the nucleotide sequences of the 40 SNP molecular markers are shown as SEQ ID No.1-SEQ ID No.40, the bases of the 40 SNP molecular markers are respectively located at the 6 th position of the sequences of SEQ ID No.1-SEQ ID No.40, and the bases of the 40 SNP molecular markers are shown as follows:

。

2. a method for identifying liquorice producing area by using SNP molecular markers for identifying liquorice produced in Xinjiang is characterized by comprising the following steps:

obtaining DNA of a sample to be detected;

carrying out library building and sequencing on the qualified sample DNA to obtain sample DNA sequencing data;

performing quality control on the sample DNA sequencing data to obtain sample DNA quality control data;

comparing the sample DNA quality control data sequence with a reference sequence, removing duplication, and carrying out SNP detection to obtain a SNP gene of a sample to be detected;

comparing the SNP genes of the sample to be detected at the same genome position with the SNP molecular marker of liquorice produced in Xinjiang in claim 1, judging whether 20 SNP genes of the sample to be detected are the same as the SNP molecular markers of liquorice produced in Xinjiang in SEQ ID NO.1-SEQ ID NO.20, and simultaneously judging whether 20 SNP genes of the sample to be detected are different from the SNP molecular markers of liquorice produced in Xinjiang in SEQ ID NO.21-SEQ ID NO. 40;

if the two judgment results are both yes, judging that the sample to be detected is the liquorice in the Xinjiang producing area; and if the result of any judgment is no, the sample to be detected is the liquorice in the non-Xinjiang producing area.

3. The method for identifying the liquorice producing area by using the SNP molecular markers for identifying liquorice produced in Xinjiang according to claim 2, wherein the sequencing of the sample DNA adopts high-throughput sequencing.

4. The method for identifying the producing area of liquorice with the SNP molecular marker for identifying liquorice produced in Xinjiang according to claim 2, wherein the quality control of the DNA sequencing data of the sample comprises the following steps:

filtering the original reads of the sample DNA sequencing data;

the filtering rules include:

removing the linker sequence;

removing reads with the number of N in the sequence being less than or equal to 2% of the length of the sequence;

reads with sequencing quality below 20 and more than 40% of the sequence length are removed.

5. The method for identifying the producing area of liquorice by using the SNP molecular marker for identifying liquorice produced in Xinjiang according to claim 2, wherein the step of comparing the DNA quality control data sequence of a sample with a reference sequence, removing the weight of the sample, and then carrying out SNP detection to obtain the SNP gene of a sample to be detected comprises the following steps:

taking a genome of liquorice in a public database as a reference sequence, and performing sequence comparison by using comparison software to obtain a bam file;

sorting the bam files by using sorting software, and removing a repeated sequence;

processing the sequenced and de-duplicated bam files by using genotyping software to obtain the preliminary SNP genotype of the sample to be detected;

and reducing the error rate of the preliminary SNP genotype of the sample by using the parameters of QUAL >100 & & FS <10 & & MQ >40 to obtain the SNP genotype of the sample to be detected.

6. An application of the SNP molecular marker of claim 1 in identifying liquorice produced in Xinjiang.

7. A gene chip or a kit comprising the SNP molecular marker of claim 1.