CN116622902B - SNP molecular marker combination for identifying wild buckwheat rhizome in northwest of Yunnan and method and application thereof - Google Patents

Abstract

The application belongs to the field of molecular biological molecular markers, and relates to a SNP molecular marker combination for identifying wild buckwheat at northwest of Yunnan and a method and application thereof, wherein the molecular marker combination comprises 40 SNP molecular markers, and the nucleotide sequences of the 40 SNP molecular markers are shown as SEQ ID No.1-SEQ ID No. 40. The SNP molecular marker combination for identifying the golden buckwheat in the northwest of Yunnan and the method and the application thereof adopt genomics technology to obtain the SNP marker on the genome level of the golden buckwheat, and can rapidly and accurately determine whether the detected golden buckwheat origin is the northwest of Yunnan.

Description

SNP molecular marker combination for identifying wild buckwheat rhizome in northwest of Yunnan and method and application thereof

Technical Field

The application belongs to the field of molecular biological molecular markers, and in particular relates to a SNP molecular marker combination for identifying wild buckwheat produced in northwest of Yunnan and a method and application thereof.

Background

The rhizoma Fagopyri Dibotryis is dried rhizome of rhizoma Fagopyri Dibotryis of Polygonaceae, also called rhizoma Fagopyri Dibotryis, the rhizoma Fagopyri Dibotryis has effects of clearing heat and detoxicating, dispelling pathogenic wind and eliminating phlegm, promoting blood circulation and removing blood stasis, invigorating spleen and promoting diuresis, and can be used for treating laryngopharynx swelling and pain, cough due to lung heat, pulmonary abscess and phlegm odor, rheumatalgia, lung abscess and dysentery.

The medicinal effect quality of the wild buckwheat at different producing areas is greatly different. Particularly to the Yunnan production place, the wild buckwheat rhizome total flavone content produced in the middle part of the Yunnan (including the places such as Kunming, jing, yuxi and the like) is high; the wild buckwheat rhizome produced in the northwest regions of Yunnan (including regions of Yangjiang, diqing, chuxiong and the like) has low content and is basically unqualified; the wild buckwheat stems produced in the south regions of Yunnan (including the regions of Wenshan, red river, and the like) are green, the old stems are slightly red, the content is medium, and the content of part of regions is qualified. The 'genuine' of the medicinal materials is that the medicinal effects of the medicinal materials are different due to the ecological environment difference of each region, and the medicinal effects can be better exerted only by specific medicinal materials planted in specific geographic climates, which is also the essence of genuine medicinal materials.

The identification of the production place of the wild buckwheat is always a technical difficulty of the identification of the wild buckwheat species, and is also an industry difficulty facing the current traditional Chinese medicine resource industry. The current methods for identifying the wild buckwheat rhizome by applying characteristics, microscopic identification and the like depend on personal experience and subjective judgment, and have low accuracy and low technical popularization.

Disclosure of Invention

Aiming at the problems, the application provides a SNP molecular marker combination for identifying wild buckwheat in northwest of Yunnan and a method and application thereof.

The application firstly provides an SNP molecular marker combination for identifying wild buckwheat at northwest of Yunnan, wherein the molecular marker combination comprises 40 SNP molecular markers, and the nucleotide sequences of the 40 SNP molecular markers are shown as SEQ ID No.1-No.40 in the application.

Further, the positions and base conditions of the 40 SNP molecular markers on the chromosome of the SNP molecular marker combination are as follows:

secondly, the application provides a method for identifying the production place of wild buckwheat by utilizing SNP molecular marker combination for identifying wild buckwheat produced in northwest of Yunnan, which is characterized by comprising the following steps:

obtaining DNA of a sample to be detected;

carrying out library-building sequencing on the DNA of the sample to be tested to obtain sample DNA sequencing data;

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

comparing the sequence of the sample DNA quality control data with a reference sequence, removing the weight, and performing SNP detection to obtain the SNP genotype of the sample to be detected;

comparing the SNP genotype of the sample to be detected at the same genome position with the SNP molecular marker combination of the golden buckwheat of the northwest part of Yunnan, judging whether the sample to be detected has 19-20 SNP molecular markers identical to the SNP molecular markers SEQ ID No.1-SEQ ID No.20 of the golden buckwheat of the northwest part of Yunnan, and judging whether the sample to be detected has 19-20 SNP molecular markers identical to the SNP molecular markers SEQ ID No. 21-SEQ ID No.40 of the golden buckwheat of the northwest part of Yunnan;

if the two judging results are yes, judging that the sample to be detected is wild buckwheat rhizome in the northwest origin of Yunnan; if the result of any judgment is negative, the sample to be detected is wild buckwheat rhizome in the non-Yunnan northwest origin.

Further, the sequencing is preceded by detecting whether the sample to be tested is qualified, and the sequencing of the sample DNA adopts 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 rule includes:

removing reads containing the linker sequence;

removing reads with the proportion of N in the reads being more than 10%;

reads with a base number ratio of less than 20 and greater than 40% were removed.

Further, comparing the sequence of the sample DNA quality control data with a reference sequence, removing the duplication and detecting the SNP to obtain the SNP genotype of the sample to be detected comprises the following steps:

taking a wild buckwheat rhizome genome from a public database as a reference sequence, and comparing the sequences by using comparison software to obtain a bam file;

sequencing the bam files by sequencing software, and removing the repeated sequence;

processing the bam file with the PCR repeated sequence removed and corrected base quality value by using genotyping software, and then obtaining the preliminary SNP genotype of the sample to be detected;

by means of "QUAL100&&FS/>10&&MQ/>And 40' parameter improves the accuracy of the sample preliminary SNP genotype to obtain the SNP genotype of the sample to be detected.

The application also provides an application of the SNP molecular marker combination in identifying the wild buckwheat produced in the northwest of Yunnan.

The application also provides a gene chip or a kit containing the SNP molecular marker combination.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions in the embodiments of the present application will be clearly and completely described in the following in conjunction with the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

Example 1 establishment of method for acquiring and identifying wild buckwheat rhizome SNP molecular markers in northwest of Yunnan

DNA extraction

DNA extraction was performed on wild buckwheat leaf material using mcAB (modified CTAB method). The specific extraction steps are as follows:

(1) Taking fresh wild buckwheat plant leaves 100 mg from the northwest part of Yunnan (Yunnan, nujiang, chuxiong, diqing) to be placed into a centrifuge tube of 2.0 mL, 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 for later use in a grinder;

(2) 1.5mL buffer A (0.1M Tris-HCl pH 8.0;5mM EDTA;0.25M NaCl;1%PVP-40) solution is added into the ground material, the centrifuge tube is repeatedly reversed to ensure that the solution and the material powder are evenly mixed, the ice bath is carried out for 10min, and the precipitated powder is evenly mixed for 3-10 times again. Centrifuging at 10000 Xg, and discarding supernatant;

(3) Repeating the step (2) once, adding 800 mu 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, homogenizing and suspending the precipitate in the solution, and then placing the solution in a 65 ℃ water bath for 1.5-2h, wherein the homogenized solution is inverted for 2-8 times;

(4) After centrifugation at 10000×g 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 10000 Xg centrifugation, carefully sucking the supernatant into a new 2.0 mL centrifuge tube with a pipette, adding an equal volume of CI solution, and mixing for 10min on a reverse shaker;

(6) After 10000 Xg centrifugation, carefully sucking the supernatant into a new 1.5mL centrifuge tube by a pipette, adding 0.6 times of ice-cold isopropanol, mixing upside down, and storing in a refrigerator at-20 ℃ for more than 1 hour;

(7) Taking out centrifuge tube in refrigerator, centrifuging at 10000×g, discarding supernatant, inverting on dry paper to drain liquid drop as much as possible, adding 100 μl RNase solution (100 mg/L), and preserving at 37deg.C for 0.5 h;

(8) Sequentially adding 150 μl ddH2O,50 μl 5M NaCl and 700 μl absolute ethanol into a centrifuge tube, mixing thoroughly, centrifuging 10000×g, discarding supernatant, and drying on paper;

(9) Adding 600 mu L of 70% ethanol, mixing the bullet bottoms, centrifuging, discarding the supernatant, and repeating the process;

(10) Ethanol was dried off in vacuo, 100 μl of TE was added to dissolve DNA, and the sample DNA concentration was determined according to NaNodrop and diluted to 50ng/μl.

2. Library-based sequencing

And (3) detecting the conventional concentration and purity of the DNA, randomly breaking the DNA sample which is qualified in detection into fragments of 350bp, and adopting a Huada self-research kit to construct a library. And after the library is qualified, performing BGI-SEQ high-throughput sequencing according to the effective concentration and the actual output requirement of the library to obtain DNA sequencing original data. The concentration detection and the purity detection can be set according to practical conditions, for example, the concentration is not lower than 50 ng/MuL, and the absorbance of the sample A260/280 is set between 1.8 and 2.0.

3. Sequencing data quality control

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

A. removing reads containing the linker sequence;

B. removing reads with the proportion of N in the reads being more than 10%;

C. reads with a base number ratio of less than 20 and greater than 40% were removed.

4. Genome assembly

Performing HiFi sequencing on a genome to obtain a high-precision sequencing sequence, and then assembling the high-precision sequencing sequence by using Hifiasm software to obtain a genome contig sequence; and then positioning the contig sequence to a chromosome by utilizing a Hi-C chromatin conformation capture technology, and finally obtaining 8 chromosome sequences of the wild buckwheat genome.

5. Data preprocessing

The genome of the golden buckwheat rhizome assembled in the previous step is used as a reference sequence, reads is compared with the reference sequence by using BWA software, and the comparison parameter is mem-t2-R. The bam files obtained by alignment were ranked using Samtools software, then PCR repeats were removed using the markdulicates tool of picard, and finally base quality values were re-corrected (BQSR).

SNP detection and screening

After the corrected bam file is obtained, the SNP genotype information gvcf file of the individual is obtained by using a Haplotypeller tool of GATK software. Merging gcvf files by using a combineGCFs tool of GATK, and then finishing mutation detection by using a GenotypeGCFs tool of GATK; to increase the accuracy of SNP detection, the parameter "QUAL" is used by VariantFilter100&&FS/>10&&MQ/>40", screening the SNP to obtain a final SNP set.

SNPs with heterozygosity greater than 0.1, mising greater than 0.1, and MAF less than 0.05 were further filtered out, and then the genetic differentiation index Fst value for each site was calculated by vcftools. Finally, 40 SNP molecular markers associated with wild buckwheat in the northwest of Yunnan are obtained through screening, the nucleotide sequences of the SNP molecular markers are shown as SEQ ID No.1-SEQ ID No.40, and the positions of the SNP molecular markers on a chromosome and the initial 10bp base sequence of 5bp before marking are shown as table 1.

TABLE 1 golden buckwheat related SNP marker information in the northwest of Yunnan

7. Establishing screening criteria

Each sample to be detected was detected based on genotypes of 40 SNP loci in Table 1. If all samples to be tested meet the detection standard of not less than 95% of the following 40 sites, the samples to be tested are the Fagopyrum cymosum in the northwest region of Yunnan, and specifically meet the following conditions: the sample to be tested has 19-20 SNP molecular markers which are the same as SNP molecular markers SEQ ID No.1-SEQ ID No.20 of the golden buckwheat of the northwest part of Yunnan, and simultaneously the sample to be tested does not have 19-20 SNP molecular markers which are the same as SNP molecular markers SEQ ID No. 21-SEQ ID No.40 of the golden buckwheat of the northwest part of Yunnan, so that the sample to be tested is the golden buckwheat of the northwest part of Yunnan, otherwise, the sample to be tested is the golden buckwheat of the northwest part of non-Yunnan.

Example 2 identification of wild buckwheat production example in northwest of Yunnan

Sample information: the wild buckwheat samples from 3 counties/villages in the south of the Yunnan, 3 counties/villages in the north-west of the Yunnan, 3 counties/villages in the middle of the Yunnan, 3 cities/counties in Guizhou province, four areas in total, and 12 specific producing areas were collected and identified according to the method and standard established in example 1.

Based on the detection results, all samples were classified and numbered for ease of analysis. Specific production place information and number information after detection of the sample are shown in table 2.

Table 2 sample sources and numbering

The identification step comprises:

1. sample DNA extraction

DNA extraction was performed on each sample of wild buckwheat leaf material using the mcAB method (modified CTAB method). The specific extraction steps are as follows:

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

(2) 1.5mL buffer A (0.1M Tris-HCl pH8.0; 5mM EDTA; 0.25M NaCl;1% PVP-40) solution was added to the milled material, the centrifuge tube was repeatedly inverted to homogenize the solution with the material powder, and the ice bath was continued for 10min, during which time the precipitated powder was again homogenized several times. Centrifuging at 10000 Xg, and discarding supernatant;

(3) Repeating the step (2) once, adding 800 mu 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, homogenizing the precipitate to suspend the solution, and then placing the solution in a 65 ℃ water bath for preserving 1.5-2h, wherein the homogenized solution is inverted for a plurality of times;

(4) After centrifugation at 10000×g 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 10000 Xg centrifugation, carefully sucking the supernatant into a new 2.0 mL centrifuge tube with a pipette, adding an equal volume of CI solution, and mixing for 10min on a reverse shaker;

(6) After 10000 Xg centrifugation, carefully sucking the supernatant into a new 1.5mL centrifuge tube by a pipette, adding 0.6 times of ice-cold isopropanol, mixing upside down, and storing in a refrigerator at-20 ℃ for more than 1 hour;

(7) Taking out centrifuge tube in refrigerator, centrifuging at 10000×g, discarding supernatant, inverting on dry paper to drain liquid drop as much as possible, adding 100 μl RNase solution (100 mg/L), and preserving at 37deg.C for 0.5 h;

(8) Sequentially adding 150 μl ddH2O,50 μl 5M NaCl and 700 μl absolute ethanol into a centrifuge tube, mixing thoroughly, centrifuging 10000×g, discarding supernatant, and drying on paper;

(9) Adding 600 mu L of 70% ethanol, mixing the bullet bottoms, centrifuging, discarding the supernatant, and repeating the process;

(10) Ethanol was dried off in vacuo, 100 μl of TE was added to dissolve DNA, and the sample DNA concentration was determined according to NaNodrop and diluted to 50ng/μl.

2. Screening and comparing to identify wild buckwheat rhizome produced in northwest of Yunnan

2.1 comparing genome SNP information of the wild buckwheat rhizome samples from each source with SNP information in Table 1, and comparing results of partial samples are shown in Table 3.

TABLE 3 SNP genotyping assay result 1

Note that: a single A or C or G or T represents homozygotes.

As shown by the identification results of Table 3, samples YWJQ101-YWJQ102, YWJQ201-YWJQ202, YWJQ301-YWJQ302 and the like all have 20 SNP molecular markers identical to those shown by SEQ ID No.1-SEQ ID No.20 of the present application, and simultaneously do not have 20 SNP molecular markers identical to those of the golden buckwheat of the northwest part of Yunnan, SEQ ID No. 21-SEQ ID No.40, while samples YSJQ101-102, YSJQ201-202, YSJQ301-302, YCJQ101-102, YCJQ 201-202, YCJQ 301-302, GZJQ101-102, GZJQ 201-202, GZJQ 301-302 and the like cannot simultaneously meet the above standards, which means that the samples YWJQ101-YWJQ102, YWJQ201-YWJQ202, YWJQ301-YWJQ302 are the northwest part of Yunnan, and the other samples are the northwest part of Yunnan, and the identification results are in accordance with the actual northwest part.

2.2 comparison results for other samples are shown in Table 4.

TABLE 4SNP genotyping assay result 2

Note that: a single A or C or G or T represents homozygotes.

As can be seen from the identification results in Table 4, the genotypes of the samples YWJQ103, YWJQ203 and YWJQ303 have 19 SNP molecular markers identical to those shown by SEQ ID No. 2-SEQ ID No.20 of golden buckwheat in the northwest of Yunnan, and have no 19 SNP molecular markers identical to those shown by SEQ ID No. 21-SEQ ID No.39, but the samples YSJQ103, YSJQ203, YSJQ303, YCJQ103, YCJQ 203, YCJQ 303, GZJQ103, GZJQ 203 and GZJQ 303 cannot meet the above standards, which means that the samples YWJQ103, YWJQ203 and YWJQ303 are the origin in the northwest of Yunnan, the other samples are the origin in the northwest of non-Yunnan, and the identification results are consistent with the practical conditions.

In summary, according to the method for identifying the origin of the golden buckwheat in the northwest of Yunnan by using 40 SNP molecular markers, the genotypes of the 40 SNP loci are compared with the genotypes of the SNPs at the same positions of the golden buckwheat to be detected, and it is determined that when 19-20 SNP molecular markers which are the same as those shown in SEQ ID No.1-SEQ ID No.20 are satisfied and 19-20 SNP molecular markers which are the same as those shown in SEQ ID No. 21-SEQ ID No.40 are not present, the sample to be detected is the golden buckwheat in the northwest of Yunnan. The identification method of the wild buckwheat rhizome in the northwest of Yunnan is simple and quick, has accurate results, and can effectively identify the wild buckwheat rhizome in the northwest of non-Yunnan.

Although the application 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 scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims (7)

1. The SNP molecular marker combination for identifying the wild buckwheat rhizome in the northwest of Yunnan is characterized in that the molecular marker consists of 40 SNP molecular markers, and the nucleotide sequence of the 40 SNP molecular markers is shown as SEQ ID No.1-SEQ ID No. 40;

the positions and base conditions of the 40 SNP molecular markers on the chromosome are as follows:

。

2. a method for identifying a wild buckwheat production site by using the SNP molecular marker set as set forth in claim 1, comprising the steps of:

obtaining DNA of a sample to be detected;

carrying out library-building sequencing on the DNA of the sample to be tested to obtain sample DNA sequencing data;

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

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

comparing the SNP genotype of the sample to be detected at the same genomic position with the SNP molecular marker combination of the wild buckwheat rhizome in the northwest of the Yunnan province in claim 1, judging whether the sample to be detected has 19-20 SNP molecular markers which are the same as those shown in SEQ ID No.1-SEQ ID No.20, and judging whether the sample to be detected has 19-20 SNP molecular markers which are the same as those shown in SEQ ID No. 21-SEQ ID No. 40;

if the two judging results are yes, judging that the sample to be detected is wild buckwheat rhizome in the northwest origin of Yunnan; if the result of any judgment is negative, the sample to be detected is wild buckwheat rhizome in the non-Yunnan northwest origin.

3. The method for identifying a place of origin of wild buckwheat according to claim 2, further characterized by detecting whether the sample to be tested is acceptable or not before said sequencing; the sequencing employs high throughput sequencing.

4. The method of claim 2, further characterized in that said quality control of sample DNA sequencing data comprises:

filtering the original reads of the sample DNA sequencing data;

the filtering rules include:

removing reads containing the linker sequence;

removing reads with the proportion of N in the reads being more than 10%;

reads with a base number ratio of less than 20 and greater than 40% were removed.

5. The method for identifying a production place of wild buckwheat rhizome according to claim 2, further characterized in that the comparing and de-duplicating the sequence of the sample DNA quality control data with a reference sequence, and performing SNP detection to obtain the SNP genotype of the sample to be detected comprises:

taking a wild buckwheat rhizome genome from a public database as a reference sequence, and carrying out sequence comparison by using comparison software bwa to obtain a bam file;

sequencing the bam files by sequencing software, and removing the repeated sequence;

processing the bam file with the PCR repeated sequence removed and corrected base quality value by using genotyping software, and then obtaining the preliminary SNP genotype of the sample to be detected;

by means of "QUAL100 && FS/>10 && MQ/>And 40' parameter improves the accuracy of the sample preliminary SNP genotype to obtain the SNP genotype of the sample to be detected.

6. The use of the SNP molecular marker combination of claim 1 for identifying cymose buckwheat production in northwest of Yunnan.

7. A kit comprising the SNP molecular marker combination of claim 1.