CN113755631B

CN113755631B - Mixed sample detection method for detecting purity of pumpkin seeds based on mSNP technology

Info

Publication number: CN113755631B
Application number: CN202111154550.5A
Authority: CN
Inventors: 许彦芬; 高苗; 刘景艺; 李凝; 张萌; 龚舒; 郝军会; 刘田; 张丛
Original assignee: Shijiazhuang Breeding Biotechnology Co ltd
Current assignee: Shijiazhuang Breeding Biotechnology Co ltd
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2024-05-24
Anticipated expiration: 2041-09-29
Also published as: CN113755631A

Abstract

The invention relates to a mixed sample detection method for detecting the purity of pumpkin seeds based on mSNP technology, which is carried out by using a primer pair 1F/R-20F/R, wherein the gene sequence of the primer pair 1F/R-20F/R is shown in SEQ ID No. 1-40; according to the invention, by adopting mSNP technology, more SNP variation can be detected under the condition that the amplicon is unchanged; by adopting the sample mixing method for detection, the sequencing cost is reduced while the amplification workload is reduced, the detection speed is also accelerated, and the detection of 1440 seeds can be finished at most in one day. The method directly reads single nucleotide polymorphism by using a sequencing technology, directly judges the purity result by a program, has visual and reliable result and avoids the influence of subjective factors in the seed development period and the result judging.

Description

Mixed sample detection method for detecting purity of pumpkin seeds based on mSNP technology

Technical Field

The invention belongs to the field of seed purity detection, and particularly relates to a mixed sample detection method for detecting the purity of pumpkin seeds based on mSNP technology.

Background

Pumpkin (Cucubita pepo l.) is an annual plant of the cucurbitaceae family, and is native to the south of north america, and is also known as zucchini. The pumpkin has both nutritive value and health care function, is popular among people, has large-area planting in various places in China, and is one of main varieties of vegetables in China. Other seeds on the market mainly depend on domestic cultivation except for a small amount of imported varieties such as French winter jade and American jade, and the seed production area is mainly concentrated in northwest of China and North of Shanxi province. The most important 2 indicators of seed quality in seed production and trade are seed purity and authenticity. The method for cultivating new hybrid varieties by utilizing the hybridization advantages is a common method for breeding at present, and false hybrid varieties often appear in the process of breeding due to human factors and the like, so that purity of the seeds is reduced, and seed purity identification is carried out by pumpkin seed production and selling units each year.

The most traditional method for identifying the purity of seeds is field planting, the harvesting time of the hybrid seeds produced by a common seed production unit is 10 late in month, and then the characteristics of plants and fruits are identified; the result of purity identification is only available after spring festival. This identification method is labor intensive, time consuming and costly. Therefore, finding a rapid, accurate and effective variety purity identification method is an urgent problem to be solved in production. With the development of biotechnology, the identification and analysis of seed purity at the DNA level is the dominant route. Wherein the molecular marker technique is a rapid and accurate seed purity identification method. Common molecular markers include RFLP, RAPD, SSR, AFLP. RFLP is a molecular marker technology invented in 1974, and has the advantages of good repeatability, co-dominance, no influence by growth and development stages and the like in seed identification; the disadvantages are insufficient information in the pedigree analysis, difficulty in identification, difficulty in operation due to the need for an isotope common detection chamber, etc. Compared with RFLP, RAPD has the advantages of less required DNA quantity, simpler operation, no need of using isotope and the like; the disadvantages are generally the inability of dominant markers to distinguish between heterozygous and homozygous, poor stability, etc. AFLP is a technology invented by Zabeau et al in the Netherlands of 1992, which can analyze crops with larger genome and polymorphism far exceeds the technologies of RFLP, RAPD and the like; the disadvantage is the need for isotopes and the like. SSR markers are co-dominant markers, so that the polymorphism is rich and the stability is good; however, it is necessary to find single copy sequences at both ends of each microsatellite at each chromosomal locus to design primers, and the difficulty of primer development is great.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a sample mixing detection method for detecting the purity of pumpkin seeds based on mSNP technology, which is efficient, accurate and low in cost.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

The technical scheme is as follows:

The primer group for detecting the purity of the pumpkin seeds in mSNP technology is characterized by comprising a primer pair 1F/R, a primer pair 2F/R, a primer pair 3F/R, a primer pair 4F/R, a primer pair 5F/R, a primer pair 6F/R, a primer pair 7F/R, a primer pair 8F/R, a primer pair 9F/R, a primer pair 10F/R, a primer pair 11F/R, a primer pair 12F/R, a primer pair 13F/R, a primer pair 14F/R, a primer pair 15F/R, a primer pair 16F/R, a primer pair 17F/R, a primer pair 18F/R, a primer pair 19F/R and a primer pair 20F/R; wherein each primer pair consists of a forward primer and a reaction primer;

In the primer pair 1F/R, the sequence of the F primer is shown as SEQ ID No.1, and the sequence of the R primer is shown as SEQ ID No. 2; in the primer pair 2F/R, the sequence of the F primer is shown as SEQ ID No.3, and the sequence of the R primer is shown as SEQ ID No. 4; in the primer pair 3F/R, the sequence of the F primer is shown as SEQ ID No.5, and the sequence of the R primer is shown as SEQ ID No. 6; in the primer pair 4F/R, the sequence of the F primer is shown as SEQ ID No.7, and the sequence of the R primer is shown as SEQ ID No. 8; in the primer pair 5F/R, the sequence of the F primer is shown as SEQ ID No.9, and the sequence of the R primer is shown as SEQ ID No. 10; in the primer pair 6F/R, the sequence of the F primer is shown as SEQ ID No.11, and the sequence of the R primer is shown as SEQ ID No. 12; in the primer pair 7F/R, the sequence of the F primer is shown as SEQ ID No.13, and the sequence of the R primer is shown as SEQ ID No. 14; in the primer pair 8F/R, the sequence of the F primer is shown as SEQ ID No.15, and the sequence of the R primer is shown as SEQ ID No. 16; in the primer pair 9F/R, the sequence of the F primer is shown as SEQ ID No.17, and the sequence of the R primer is shown as SEQ ID No. 18; in the primer pair 10F/R, the sequence of the F primer is shown as SEQ ID No.19, and the sequence of the R primer is shown as SEQ ID No. 20; in the primer pair 11F/R, the sequence of the F primer is shown as SEQ ID No.21, and the sequence of the R primer is shown as SEQ ID No. 22; in the primer pair 12F/R, the sequence of the F primer is shown as SEQ ID No.23, and the sequence of the R primer is shown as SEQ ID No. 24; in the primer pair 13F/R, the sequence of the F primer is shown as SEQ ID No.25, and the sequence of the R primer is shown as SEQ ID No. 26; in the primer pair 14F/R, the sequence of the F primer is shown as SEQ ID No.27, and the sequence of the R primer is shown as SEQ ID No. 28; in the primer pair 15F/R, the sequence of the F primer is shown as SEQ ID No.29, and the sequence of the R primer is shown as SEQ ID No. 30; in the primer pair 16F/R, the sequence of the F primer is shown as SEQ ID No.31, and the sequence of the R primer is shown as SEQ ID No. 32; in the primer pair 17F/R, the sequence of the F primer is shown as SEQ ID No.33, and the sequence of the R primer is shown as SEQ ID No. 34; in the primer pair 18F/R, the sequence of the F primer is shown as SEQ ID No.35, and the sequence of the R primer is shown as SEQ ID No. 36; in the primer pair 19F/R, the sequence of the F primer is shown as SEQ ID No.37, and the sequence of the R primer is shown as SEQ ID No. 38;

In the primer pair 20F/R, the sequence of the F primer is shown as SEQ ID No.39, and the sequence of the R primer is shown as SEQ ID No. 40.

Further, the primer pair 1F/R-20F/R is obtained by mSNP technology.

The second technical scheme is as follows:

a mixed sample detection method for detecting the purity of pumpkin seeds according to the primer group comprises the following steps:

step1, selecting materials: selecting 1 or more pumpkin varieties; at least 96 seeds are adopted for each pumpkin sample;

step 2, accurately quantifying the pumpkin genome DNA;

Step 3, synthesizing primers in the primer groups, wherein 10 primers with different target labels are synthesized when the forward primer and the reverse primer in each primer pair are synthesized; then mixing primers according to the appointed label combination to prepare primer mixed solution;

Step 4, performing one-round PCR amplification on the genome DNA of the pumpkin by using the genome DNA of the pumpkin as a template and using the primer mixture to obtain a target region;

step 5, mixing the obtained PCR amplification products in equal amounts;

step6, screening fragments of the mixed products;

Step 7, digesting the single-stranded DNA in the system obtained after screening;

Step 8, purifying the digested product;

step 9, configuring a two-round PCR system in the system obtained in the step 8;

step 10, purifying the two rounds of PCR products to finish the preparation of a sequencing library;

step 11, mixing the sequencing library with the same mass, and then sequencing by a machine to obtain sequencing data;

step 12, disconnecting the obtained test data again according to the label combination;

and 13, identifying the genotype result of the target site of the test sample, and judging the purity of the seeds according to the genotype condition of the site.

Further, when the forward primer and the reverse primer in the primer pair are synthesized, 10 primers with different target labels are synthesized;

in the tag combination of each primer pair, the tag sequence of the forward primer is different from the tag sequence of the reverse primer.

Further, in the step 3, the tag sequences in the synthesized 10 forward primers with tags in each primer pair are respectively shown as SEQ ID No. 45-54;

The tag sequences in the synthesized 10 tagged reverse primers are shown in SEQ ID Nos. 55-64, respectively, for each primer pair.

Further, 10 pairs of tagged primers were synthesized for each primer pair, and the manner of tag combination of the forward primer and the reverse primer was selected from any one or more of Table 1.

Table 1: label combination mode

Further, in the step 3, the F primer in the primer pair 1F/R-20F/R further comprises an F-terminal universal primer, and the sequence of the F-terminal universal primer is shown as SEQ ID No. 41; the R primer in the primer pair 1F/R-20F/R also comprises an R-terminal universal primer, and the sequence of the R-terminal universal primer is shown as SEQ ID No. 42;

The sequence of Pr imer F used in the two rounds of PCR in the step 9 is shown as SEQ ID No.43,

The sequence of Pr imer R used is shown in SEQ ID No. 44.

Still further, when the number of zucchini varieties is plural, the Pr imer R sequences further include a barcode sequence for distinguishing the zucchini varieties.

Further, in step 2, in the tag combination of each primer pair, the tag sequence of the forward primer is different from the tag sequence of the reverse primer.

Further, in step 1, the genomic DNA of the pumpkin seeds is extracted using a pumpkin seed genomic DNA extraction kit.

Further, in step 4, the round of PCR amplification system: 8 μl of primer mix; the DNA dosage is 100ng;3*T enzyme 15. Mu.l; adding water to make up 45 μl;

The round of PCR amplification procedure: 3min at 95 ℃; (95 ℃ C. 30s,60 ℃ C. 4min,72 ℃ C. 30 s) 28 cycles; and at 72℃for 4min.

Further, in step6, the mixed product is subjected to fragment screening, which comprises the following specific operations:

step 6.1, adding a round of magnetic beads with the volume of 0.4 times of that of the PCR, blowing and mixing uniformly up and down by using a pipettor, standing for 2min, adsorbing by using a magnetic frame until the solution is clarified, and transferring the supernatant into a new tube;

Step 6.2, adding a round of magnetic beads with the volume of 0.6 times of that of the PCR, blowing and mixing uniformly up and down by using a pipettor, standing for 2min, adsorbing by using a magnetic frame until the solution is clarified, and removing the supernatant;

step 6.3, adding a round of magnetic bead suspension with the volume of 0.9 times of that of the PCR, re-suspending the magnetic beads, standing for 2min, adsorbing by a magnetic frame until the solution is clear, and removing the supernatant;

and 6.4, adding 100 mu l of 80% ethanol, repeatedly adsorbing the magnetic beads on different two sides by using a magnetic rack, fully washing the magnetic beads, adsorbing for 2min by using the magnetic rack, removing the supernatant, and standing at room temperature until the ethanol volatilizes cleanly.

In the step 7, single-stranded DNA in the system obtained after screening is digested, and the specific operation steps are as follows:

step 7.1, adding 20 mu l of water into the obtained product, and uniformly mixing the magnetic beads;

step 7.2, adsorbing magnetic beads, transferring 16 μl of supernatant to a new EP tube;

Step 7.3, adding 2 μl of Exo I and 2 μl of 10×reaction Buffer into the system;

and 7.4, the digestion procedure of the digestion system is as follows: 30min at 37 ℃; 15min at 85 ℃;

in step 8, the specific operation steps of purifying the digested product are as follows:

8.1, adding magnetic beads of which the number is 0.9, blowing and mixing uniformly up and down by using a pipettor, standing for 2min, adsorbing by using a magnetic frame until the solution is clear, and removing the supernatant;

8.2, adding a magnetic bead heavy suspension with the same PCR volume, re-suspending the magnetic beads, standing for 2min, adsorbing by a magnetic frame until the solution is clear, and removing the supernatant;

And 8.3, adding 100 mu l of 80% ethanol, repeatedly adsorbing magnetic beads on different sides by using a magnetic rack, fully washing the magnetic beads, adsorbing for 2min by using the magnetic rack, removing the supernatant, and standing at room temperature until the ethanol volatilizes cleanly.

Further, in step 9, the two-round PCR system: 3*T enzyme 10. Mu.l; pr imer F; pr imer R; H2O18 μl;

The two-round PCR procedure: 3min at 95 ℃; (95 ℃ 15s,58 ℃ 15s,72 ℃ 30 s) 12 cycles; 72 ℃ for 4min;

Further, in step 10, the two rounds of PCR products were purified using 0.80 times of magnetic beads, and the specific procedure was as follows:

step 10.1, adding magnetic beads of which the number is 0.8, blowing and mixing uniformly up and down by using a pipettor, standing for 2min, adsorbing by using a magnetic frame until the solution is clear, and removing the supernatant;

step 10.2, adding a magnetic bead heavy suspension with the same PCR volume, re-suspending the magnetic beads, standing for 2min, adsorbing by a magnetic frame until the solution is clear, and removing the supernatant;

Step 10.3, adding 100 μl of 80% ethanol, repeatedly adsorbing magnetic beads on different sides by using a magnetic rack, sufficiently washing the magnetic beads, adsorbing for 2min by using the magnetic rack, removing the supernatant, and standing at room temperature until the ethanol volatilizes completely;

step 10.4, adding 23 mu l E l ut ion Buffer, fully suspending magnetic beads, standing at room temperature for 2min to elute DNA, adsorbing the magnetic beads by using a magnet, and sucking the obtained supernatant DNA solution into a new tube to obtain a sequencing library; the E l ut ionBuffer is 10mM Tri s-HCl, pH 8.0-8.5.

Compared with the prior art, the invention has the following beneficial effects:

mSNP technology: the invention adopts mSNP technology, a plurality of SNP are corresponding in one amplicon, the information obtained by each amplicon fragment is utilized to the maximum extent, and SNP variation can be detected as much as possible under the condition that the amplicon is unchanged. And haplotypes can be formed among the SNPs, so that the mutation detection efficiency is improved. Therefore, not only can the variation in mSNP loci and among loci be adopted, but also the detection can be carried out by adopting a haplotype and SNP modes, so that the detection of the genetic variation is finer, and the accuracy and the sensitivity of the marker identification are improved. In the patent, 20 pairs of primer pairs are adopted, and the actually detected mutation information is more than 100, so that more polymorphic sites meeting the requirements can be screened out for purity identification analysis. Compared with the conventional SNP detection, the primer pair number is reduced, and the cost is reduced.

The cost is low: by adopting mSNP technology, more SNP variations can be detected under the condition that the amplicon is unchanged; by adopting a scheme of mixing and detecting at least 96 test sample products after one round of amplification, the sequencing cost is reduced while the workload of two rounds of amplification is reduced.

High efficiency: by adopting the mixed sample detection scheme, at least 96 products of detection samples are mixed for subsequent amplification and sequencing after one round of amplification, and the detection of 1440 seeds can be finished at most in one day. Compared with other detection methods, the method is simple to operate, does not need field planting, does not need experimental operation with large workload and large detection difficulty, and can rapidly detect seed purity.

The accuracy is that: the method directly reads single nucleotide polymorphism by using a sequencing technology, directly judges the purity result by a program, has visual and reliable result and avoids the influence of subjective factors in the seed development period and the result judging.

Drawings

FIG. 1 is a flow chart of the method for detecting mixed samples according to the present invention.

Detailed Description

Example 1: method for obtaining specific primers

The method for obtaining the specific primer comprises the following steps:

The whole genome resequencing data of the pumpkin is utilized, BWA-mem (http:// bio-BWA. Sourceforge. Net /) is adopted to be pasted back to the pumpkin reference genome, and GATK (https:// software. Broad inst. Org/GATK /) is utilized for single nucleotide variation identification.

The identified single nucleotide variation locus set is screened, the minimum allelic variation frequency is more than 0.02, the heterozygosity rate is less than 15 percent, the deletion rate is less than 20 percent, single nucleotide variation loci are combined, a section with the single nucleotide locus number of 6-9 is screened, namely mSNP (polymononucleotide polymorphism) loci, compared with the traditional SNP (single nucleotide polymorphism) loci, mSNP loci can maximally utilize the information obtained by each primer pair, namely, as many SNP loci as possible are detected under the condition that the primer pair is unchanged, and all SNPs in the same primer pair can be combined into haplotypes, so that the polymorphism is higher. For example, there are two variants of A/T in one SNP, and the distinguishable polymorphisms are AA, AT, TT, 3 in total, if mSNP sites are detected, if there are 3 SNPs (A/T, G/T, C/A) in one primer pair, 8 (AGC, AGA, ATA, ATC, TGC, TGA, TTA, TTC) polymorphisms can be present. This allows for finer detection of genetic variations while improving the accuracy and sensitivity of marker identification. The number of the segments with high polymorphism screening is 40 in total.

The primer design is carried out on 40 target segments, and the primer specificity is screened, so that a total of 131 mononucleotide variation sites of 20 pairs of chromosome specific primers are obtained. From the comprehensive consideration of detection cost and practical angle, 20 pairs of primer mixtures are finally selected according to the principle of 5-10% diversity among pumpkin samples, and the purity of pumpkin seeds is detected, wherein about 120 SNP loci can be detected in total.

In the invention, 20 groups of specific primer pairs are obtained in total, namely primer pairs 1F/R-20F/R, and the gene sequences of the primer pairs 1F/R-20F/R are shown as SEQ ID No. 1-40.

Example 2: primer group for detecting purity of pumpkin seeds

The primer group for detecting the purity of the pumpkin seeds comprises a primer pair 1F/R-20F/R, wherein the primer pair 1F/R-20F/R not only comprises a specific primer sequence shown as SEQ ID No. 1-40, but also comprises a universal primer sequence, and the F end universal primer sequence of the F primer in the primer pair 1F/R-20F/R is shown as SEQ ID No. 41; the sequence of the universal primer at the R end of the R primer in the primer pair 1F/R-20F/R is shown as SEQ ID No. 42;

example 3: the method for obtaining the primer mixture comprises the following steps:

After the specific primer is obtained, a specific tag sequence is designed, and then primer synthesis is performed again, wherein the specific tag sequence is added during the primer synthesis, and 96 groups of specific tag combinations are adopted in the embodiment, specifically:

according to the specific label combination condition, synthesizing 10 primers with different target labels from each specific primer, wherein the sequence form of the primers with the target labels is shown in table 2, and taking 10 μl of each primer to 10ml; the concentration of each primer is 0.1 mu M, and the primer pair containing 96 groups of specific tag combinations, namely primer mixed solution, is prepared in total in the embodiment;

TABLE 2 primer set

Primer pair	F Forward primer (5 '-3')	R reverse primer (5 '-3')
			1F/R	FFFYYYCATGCTACTGGTGCTTCAAAGTATT	RRRYYYGCATAGGGCAAAAATCCTAGCATAT
2F/R	FFFYYYGACTTGAAGTCAACGTTTGTTCAGA	RRRYYYTTGATGATGAAATGGGAGGAGTCAT
			3F/R	FFFYYYAAGAAAACCCAAAACATTATATCATAAACT	RRRYYYTGGAAACTACTTCATATAAGACGCG
4F/R	FFFYYYGTTGCTTCGCCTTCGTTCTTC	RRRYYYTCTCAAAGATTTCATGCCATCACAG
			5F/R	FFFYYYACATTAGCTCCAAGTGATTGATTGT	RRRYYYACTCCAGACTATGCAATTCTGACTT
6F/R	FFFYYYGAGATTCAGATGGCTGGGAAATAGG	RRRYYYCCACAAACTACTCACACCAGAATTC
			7F/R	FFFYYYCCGATCTGGATATCTCACCTGAAAT	RRRYYYGAGTTGGTTGGCATCTCCTCTTA
8F/R	FFFYYYATTCAACCATCACTTCATTTCCTCG	RRRYYYTTGGTGAAGAACAAGAGAGAGAGAG
			9F/R	FFFYYYCTCTCGAGCTACATGAATCAAGAGA	RRRYYYCCCTGAATTTGAATACTCGTGTAGC
10F/R	FFFYYYCTATTATGCCGCAGCTCAACTTAAA	RRRYYYGATGAAGAACACAGTTCCAAAGAGG
			11F/R	FFFYYYACTGTGCGGTGTGTCAGC	RRRYYYCAATCATCAGGCTTATATGGTGCTC
12F/R	FFFYYYGGTGCAGAGTTTAGATGGAAAGAAC	RRRYYYGGACAGAGAAGTATGGGAAAGCATT
			13F/R	FFFYYYATGTCATAAGTTCTCTTGCCTGACA	RRRYYYTTGTTAGAAAAAGCATTAGAGCCGG
14F/R	FFFYYYGAATTTTAAGATTATGTGCACAGTTGT	RRRYYYTGTTTACCAAGGGAAACATCCAAAG
			15F/R	FFFYYYCTTAAGAAATCGACAAAGTGCTTCG	RRRYYYGCCTTCCCTAAAATTCCAAACAATG
16F/R	FFFYYYGATAGCCATGGTTGCACTAATGAAA	RRRYYYGTTTTTCCCTCTTCTGTCTTGGAAA
			17F/R	FFFYYYCTAACACACCGCTCAATGTCTAAC	RRRYYYGGATGATGTGAAAGAGGAATTGTGA
18F/R	FFFYYYATCCCACATTTGTTGGATAGGAGAA	RRRYYYATCTCTCCATTGTCTATCCCGATTG
			19F/R	FFFYYYAGAAGTAGTTTTCAAGAACCCATTTT	RRRYYYCATCAACCTGGAATGATGGTCAAAG
20F/R	FFFYYYCGGATGATCCACTCACATTTTTCTT	RRRYYYTAGATTTAGATGATGCAGGCCAACC

"FFF" is the F-terminal universal primer sequence AACGACATGGCTACGATCCGACTT, shown as SEQ ID No. 41;

the RRR is an R-end universal primer sequence, and the R-end universal primer sequence is CTAAGACCGCTTGGCCTCCGACTT as shown in SEQ ID No. 42.

Wherein "YYY" is a tag sequence,

The label sequences in the synthesized 10 forward primers with labels in each primer pair are CCTTC, ACCGA, ATGTG, AATGC, TTCGG, AAGGT, CCCAT, ATGGA, ACGAT, CTCTG respectively, namely shown as SEQ ID No. 45-54 respectively;

each primer pair has a label sequence ATCCG, TATCG, ACTCG, TAACC, CTTAC, TCCTA, ACACT, TACGT, TCACG, ACGCA in the synthesized 10 reverse primers with labels, namely shown in SEQ ID Nos. 55-64.

Each primer pair synthesizes 10 primer pairs with labels, and the label combination mode of the forward primer and the reverse primer is selected from any one or a plurality of the primer pairs in the table 2.

Example 4: pure seed identification

Step 1, selecting materials, namely selecting 2 parts of pumpkin (96 seeds are selected for each part of pumpkin respectively), respectively numbering CF-11 and CF-15, extracting genome DNA of the pumpkin seeds, and accurately quantifying the extracted DNA by adopting a pumpkin seed genome DNA extraction kit produced by Shijizhuang Boruidi biotechnology Co.

Step2, performing one round of PCR amplification by using pumpkin seed genome DNA as a template and using a primer mixed solution to obtain a target region;

the round of PCR amplification system: 8. Mu.l of the primer mixture obtained in example 3; the DNA dosage is 100ng;3*T enzyme 15. Mu.l; water was added to make up 45. Mu.l.

And 3, mixing the obtained PCR amplification products in equal amounts, wherein the amplification products can be mixed only by using specific labels with different combinations, and the mixing can be directly carried out according to a system 1:1. After equal amount mixing, purifying the mixed product, namely screening fragments, wherein the specific steps are as follows;

Step 3.1, adding a round of magnetic beads with the volume of 0.4 times of that of the PCR, blowing and mixing uniformly up and down by using a pipettor, standing for 2min, adsorbing by using a magnetic frame until the solution is clarified, and transferring the supernatant into a new tube;

step 3.2, adding a round of magnetic beads with the volume of 0.6 times of that of the PCR, blowing and mixing uniformly up and down by using a pipettor, standing for 2min, adsorbing by using a magnetic frame until the solution is clarified, and removing the supernatant;

step 3.3, adding a round of magnetic bead suspension with the volume of 0.9 times of the PCR volume, re-suspending the magnetic beads, standing for 2min, adsorbing by a magnetic rack until the solution is clear, and removing the supernatant;

Step 3.4, 100 μl of 80% ethanol is added, and the magnetic beads are repeatedly adsorbed on different sides by using a magnetic rack, so that the magnetic beads are sufficiently washed. Adsorbing with a magnetic rack for 2min, removing supernatant, and standing at room temperature until ethanol volatilizes completely;

the magnetic beads are as follows: nuance magnetic bead

Step 4, digesting the single-stranded DNA in the system obtained after screening;

in the magnetic bead-containing system obtained in step 3, the following operations were performed:

Step 4.1, adding 20 mu l of water into the obtained product, and uniformly mixing the magnetic beads;

step 4.2, adsorbing magnetic beads, transferring 16 μl of supernatant to a new EP tube;

step 4.3, add 2. Mu.l Exo I, 10*React ion Buffer 2. Mu.l to the above system.

Step 4.4, the digestion procedure of the system is as follows: 30min at 37 ℃; 15min at 85 ℃.

Step 5, purifying the digested product:

step 5.1, adding magnetic beads of which the number is 0.9, blowing and mixing uniformly up and down by using a pipettor, standing for 2min, adsorbing by using a magnetic frame until the solution is clear, and removing the supernatant;

step 5.2, adding a magnetic bead heavy suspension with the same PCR volume, re-suspending the magnetic beads, standing for 2min, adsorbing by a magnetic frame until the solution is clear, and removing the supernatant;

And 5.3, adding 100 mu l of 80% ethanol, and repeatedly adsorbing the magnetic beads on different two sides by using a magnetic rack to sufficiently wash the magnetic beads. Adsorbing with a magnetic rack for 2min, removing supernatant, and standing at room temperature until ethanol volatilizes completely.

Step 6, configuring a two-round PCR system in the system obtained in the step 5:

Configuring a two-round PCR system in the system containing the magnetic beads obtained in the step 5, and carrying out two-round PCR amplification;

the two-round PCR system: 3*T enzyme 10. Mu.l; pr imer F; pr imer R; h ₂ O18 μl

The two-round PCR procedure: 3min at 95 ℃; (95 ℃ 15s,58 ℃ 15s,72 ℃ 30 s) 12 cycles; and at 72℃for 4min.

The sequence of Pr imer F is shown as SEQ ID No.43 and is GAACGACATGGCTACGATCCGACTT; the sequence of Pr imerR is shown as SEQ ID No.44 and is TGTGAGCCAAGGAGTTGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTT;

Because 2 zucchini varieties are adopted in the embodiment, in order to distinguish samples, the sequence Pr imer R comprises a unique Barcode sequence Barcode in addition to the sequence shown as SEQ ID No. 44;

The sequence of Pr imer R with bar code is:

TGTGAGCCAAGGAGTTGxxxxxxxxxxTTGTCTTCCTAAGACCGCTTGGCCTCCGACTT；

where "xxxxxxx" is the unique Barcode used to identify the sample to distinguish the sample.

In the embodiment, the Barcode sequences of 2 pumpkin variety samples are CGGCTAAA, TCCCCGTG respectively; namely as shown in SEQ ID Nos. 65-66, respectively.

Step 7, purifying the two-round PCR amplified product by using 0.80 times of magnetic beads to finish the preparation of a sequencing library;

step 7.1, adding magnetic beads of which the number is 0.8, blowing and mixing the magnetic beads up and down by using a pipettor, standing for 2min, adsorbing the magnetic beads by using a magnetic rack until the solution is clarified, and removing the supernatant;

step 7.2, adding a magnetic bead heavy suspension with the same PCR volume, re-suspending the magnetic beads, standing for 2min, adsorbing by a magnetic frame until the solution is clear, and removing the supernatant;

Step 7.3, 100 μl of 80% ethanol is added, and the magnetic beads are repeatedly adsorbed on different sides by using a magnetic rack, so that the magnetic beads are sufficiently washed. Adsorbing with a magnetic rack for 2min, removing supernatant, and standing at room temperature until ethanol volatilizes completely.

Step 7.4, 23 mu l E l ut ion Buffer was added, the beads were sufficiently suspended, and the mixture was allowed to stand at room temperature for 2min to elute DNA. Adsorbing the magnetic beads by using a magnet, and sucking the obtained supernatant DNA solution into a new tube to obtain a sequencing library (E l ut ion Buffer is 10mM Tri s-HCl, pH 8.0-8.5);

and 8, mixing the sequencing library with the same mass, and then performing on-machine sequencing to obtain sequencing data.

Step 9, identifying the genotype result of the target DNA, respectively selecting the seeds synthesized by detection from 96 seeds for subsequent purity judgment, and judging the purity of the seeds according to the locus genotype condition with high polymorphism, wherein the detection result of the embodiment is shown in Table 3.

TABLE 3 detection results

Sample numbering	Number of selfed seeds	Seed purity	Conclusion(s)
				CF-11	2	97.85％	Same as the conventional identification result
CF-15	3	96.84％	Same as the conventional identification result

Seed purity program interpretation principle:

Firstly, judging whether each locus is hybridized/outcrossed or selfed, and judging the standard: the proportion of the genotype species of a batch of seeds at each locus is calculated. If the genotype of this locus is only one and homozygous, this locus is discarded because it cannot be determined whether it is self-mating or hybrid. If the proportion of one heterozygous genotype exceeds 90%, judging as hybridization; the genotype of a sample at this site is homozygous and judged to be selfing; if other heterozygous genotypes are identified as outcrossing.

The samples were then submitted for selfing, outcrossing and hybridization. And finally, counting the whole condition of the batch of seeds.

Purity calculation, expressed as a percentage of seed purity:

The above described embodiments are only preferred examples of the invention and are not exhaustive of the possible implementations of the invention. Any obvious modifications thereof, which would be apparent to those skilled in the art without departing from the principles and spirit of the present invention, should be considered to be included within the scope of the appended claims.

Sequence listing

<110> Shijia Boruidi Biotechnology Co., ltd

<120> A mixed sample detection method for detecting the purity of pumpkin seeds based on mSNP technology

<130> 4

<160> 66

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 1

catgctactg gtgcttcaaa gtatt 25

<210> 2

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 2

gcatagggca aaaatcctag catat 25

<210> 3

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 3

gacttgaagt caacgtttgt tcaga 25

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 4

ttgatgatga aatgggagga gtcat 25

<210> 5

<211> 30

<212> DNA

<213> Artificial sequence (unknown)

<400> 5

aagaaaaccc aaaacattat atcataaact 30

<210> 6

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 6

tggaaactac ttcatataag acgcg 25

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence (unknown)

<400> 7

gttgcttcgc cttcgttctt c 21

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 8

tctcaaagat ttcatgccat cacag 25

<210> 9

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 9

acattagctc caagtgattg attgt 25

<210> 10

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 10

actccagact atgcaattct gactt 25

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 11

gagattcaga tggctgggaa atagg 25

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 12

ccacaaacta ctcacaccag aattc 25

<210> 13

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 13

ccgatctgga tatctcacct gaaat 25

<210> 14

<211> 23

<212> DNA

<213> Artificial sequence (unknown)

<400> 14

gagttggttg gcatctcctc tta 23

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 15

attcaaccat cacttcattt cctcg 25

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 16

ttggtgaaga acaagagaga gagag 25

<210> 17

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 17

ctctcgagct acatgaatca agaga 25

<210> 18

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 18

ccctgaattt gaatactcgt gtagc 25

<210> 19

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 19

ctattatgcc gcagctcaac ttaaa 25

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 20

gatgaagaac acagttccaa agagg 25

<210> 21

<211> 18

<212> DNA

<213> Artificial sequence (unknown)

<400> 21

actgtgcggt gtgtcagc 18

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 22

caatcatcag gcttatatgg tgctc 25

<210> 23

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 23

ggtgcagagt ttagatggaa agaac 25

<210> 24

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 24

ggacagagaa gtatgggaaa gcatt 25

<210> 25

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 25

atgtcataag ttctcttgcc tgaca 25

<210> 26

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 26

ttgttagaaa aagcattaga gccgg 25

<210> 27

<211> 27

<212> DNA

<213> Artificial sequence (unknown)

<400> 27

gaattttaag attatgtgca cagttgt 27

<210> 28

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 28

tgtttaccaa gggaaacatc caaag 25

<210> 29

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 29

cttaagaaat cgacaaagtg cttcg 25

<210> 30

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 30

gccttcccta aaattccaaa caatg 25

<210> 31

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 31

gatagccatg gttgcactaa tgaaa 25

<210> 32

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 32

gtttttccct cttctgtctt ggaaa 25

<210> 33

<211> 24

<212> DNA

<213> Artificial sequence (unknown)

<400> 33

ctaacacacc gctcaatgtc taac 24

<210> 34

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 34

ggatgatgtg aaagaggaat tgtga 25

<210> 35

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 35

atcccacatt tgttggatag gagaa 25

<210> 36

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 36

atctctccat tgtctatccc gattg 25

<210> 37

<211> 26

<212> DNA

<213> Artificial sequence (unknown)

<400> 37

agaagtagtt ttcaagaacc catttt 26

<210> 38

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 38

catcaacctg gaatgatggt caaag 25

<210> 39

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 39

cggatgatcc actcacattt ttctt 25

<210> 40

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 40

tagatttaga tgatgcaggc caacc 25

<210> 41

<211> 24

<212> DNA

<213> Artificial sequence (unknown)

<400> 41

aacgacatgg ctacgatccg actt 24

<210> 42

<211> 24

<212> DNA

<213> Artificial sequence (unknown)

<400> 42

ctaagaccgc ttggcctccg actt 24

<210> 43

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<400> 43

gaacgacatg gctacgatcc gactt 25

<210> 44

<211> 49

<212> DNA

<213> Artificial sequence (unknown)

<400> 44

tgtgagccaa ggagttgttg tcttcctaag accgcttggc ctccgactt 49

<210> 45

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 45

ccttc 5

<210> 46

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 46

accga 5

<210> 47

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 47

atgtg 5

<210> 48

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 48

aatgc 5

<210> 49

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 49

ttcgg 5

<210> 50

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 50

aaggt 5

<210> 51

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 51

cccat 5

<210> 52

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 52

atgga 5

<210> 53

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 53

acgat 5

<210> 54

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 54

ctctg 5

<210> 55

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 55

atccg 5

<210> 56

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 56

tatcg 5

<210> 57

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 57

actcg 5

<210> 58

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 58

taacc 5

<210> 59

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 59

cttac 5

<210> 60

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 60

tccta 5

<210> 61

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 61

acact 5

<210> 62

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 62

tacgt 5

<210> 63

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 63

tcacg 5

<210> 64

<211> 5

<212> DNA

<213> Artificial sequence (unknown)

<400> 64

acgca 5

<210> 65

<211> 8

<212> DNA

<213> Artificial sequence (unknown)

<400> 65

cggctaaa 8

<210> 66

<211> 8

<212> DNA

<213> Artificial sequence (unknown)

<400> 66

tccccgtg 8

Claims

1. The primer group for detecting the pumpkin seed purity based on mSNP technology is characterized by comprising a primer pair 1F/R, a primer pair 2F/R, a primer pair 3F/R, a primer pair 4F/R, a primer pair 5F/R, a primer pair 6F/R, a primer pair 7F/R, a primer pair 8F/R, a primer pair 9F/R, a primer pair 10F/R, a primer pair 11F/R, a primer pair 12F/R, a primer pair 13F/R, a primer pair 14F/R, a primer pair 15F/R, a primer pair 16F/R, a primer pair 17F/R, a primer pair 18F/R, a primer pair 19F/R and a primer pair 20F/R; wherein each primer pair consists of a forward primer and a reaction primer;

In the primer pair 1F/R, the sequence of the F primer is shown as SEQ ID No.1, and the sequence of the R primer is shown as SEQ ID No. 2;

In the primer pair 2F/R, the sequence of the F primer is shown as SEQ ID No.3, and the sequence of the R primer is shown as SEQ ID No. 4;

In the primer pair 3F/R, the sequence of the F primer is shown as SEQ ID No.5, and the sequence of the R primer is shown as SEQ ID No. 6;

In the primer pair 4F/R, the sequence of the F primer is shown as SEQ ID No.7, and the sequence of the R primer is shown as SEQ ID No. 8;

In the primer pair 5F/R, the sequence of the F primer is shown as SEQ ID No.9, and the sequence of the R primer is shown as SEQ ID No. 10;

In the primer pair 6F/R, the sequence of the F primer is shown as SEQ ID No.11, and the sequence of the R primer is shown as SEQ ID No. 12;

In the primer pair 7F/R, the sequence of the F primer is shown as SEQ ID No.13, and the sequence of the R primer is shown as SEQ ID No. 14;

in the primer pair 8F/R, the sequence of the F primer is shown as SEQ ID No.15, and the sequence of the R primer is shown as SEQ ID No. 16;

in the primer pair 9F/R, the sequence of the F primer is shown as SEQ ID No.17, and the sequence of the R primer is shown as SEQ ID No. 18;

in the primer pair 10F/R, the sequence of the F primer is shown as SEQ ID No.19, and the sequence of the R primer is shown as SEQ ID No. 20;

In the primer pair 11F/R, the sequence of the F primer is shown as SEQ ID No.21, and the sequence of the R primer is shown as SEQ ID No. 22;

In the primer pair 12F/R, the sequence of the F primer is shown as SEQ ID No.23, and the sequence of the R primer is shown as SEQ ID No. 24;

in the primer pair 13F/R, the sequence of the F primer is shown as SEQ ID No.25, and the sequence of the R primer is shown as SEQ ID No. 26;

in the primer pair 14F/R, the sequence of the F primer is shown as SEQ ID No.27, and the sequence of the R primer is shown as SEQ ID No. 28;

in the primer pair 15F/R, the sequence of the F primer is shown as SEQ ID No.29, and the sequence of the R primer is shown as SEQ ID No. 30;

in the primer pair 16F/R, the sequence of the F primer is shown as SEQ ID No.31, and the sequence of the R primer is shown as SEQ ID No. 32;

in the primer pair 17F/R, the sequence of the F primer is shown as SEQ ID No.33, and the sequence of the R primer is shown as SEQ ID No. 34;

In the primer pair 18F/R, the sequence of the F primer is shown as SEQ ID No.35, and the sequence of the R primer is shown as SEQ ID No. 36;

In the primer pair 19F/R, the sequence of the F primer is shown as SEQ ID No.37, and the sequence of the R primer is shown as SEQ ID No. 38;

2. A mixed sample detection method for detecting the purity of pumpkin seeds by using the primer group according to claim 1, which comprises the following steps:

step 1, selecting materials: selecting 1 or more pumpkin varieties; at least 96 seeds are adopted for each pumpkin sample;

step 2, accurately quantifying the pumpkin genome DNA;

step 3, synthesizing primers in the primer groups in claim 1, wherein 10 primers with different target labels are synthesized when the forward primer and the reverse primer in each primer pair are synthesized; then mixing primers according to the appointed label combination to prepare primer mixed solution;

step 5, mixing the obtained PCR amplification products in equal amounts;

step6, screening fragments of the mixed products;

Step 8, purifying the digested product;

3. The method for detecting a mixed sample according to claim 2, wherein,

In the step 3, the F primer in the primer pair 1F/R-20F/R further comprises an F-terminal universal primer, and the sequence of the F-terminal universal primer is shown as SEQ ID No. 41; the R primer in the primer pair 1F/R-20F/R also comprises an R-terminal universal primer, and the sequence of the R-terminal universal primer is shown as SEQ ID No. 42;

In the step 9, the sequence of the Primer F used in the two rounds of PCR is shown as SEQ ID No. 43; the sequence of Primer R used in the two rounds of PCR is shown as SEQ ID No. 44.

4. The method for detecting a mixed sample according to claim 3, wherein,

When the number of the pumpkin varieties is plural, the sequence of the Primer R further comprises a barcode sequence for distinguishing the pumpkin varieties.

5. The method according to claim 2, wherein in step 2, the tag sequence of the forward primer is different from the tag sequence of the reverse primer in the tag combination of each primer pair.

6. The method for detecting a mixed sample according to claim 2, wherein,

In the step1, the genome DNA of the pumpkin seeds is extracted by adopting a pumpkin seed genome DNA extraction kit.

7. The method for detecting a mixed sample according to claim 2, wherein,

In step 4, the round of PCR amplification procedure: 3min at 95 ℃;95 ℃ for 30s,60 ℃ for 4min,72 ℃ for 30s,28 cycles; and at 72℃for 4min.

8. The method for detecting a mixed sample according to claim 2, wherein,

In step 6, the mixed product is subjected to fragment screening, and the specific operation is as follows:

step 6.4, adding 100 mu l of 80% ethanol, repeatedly adsorbing magnetic beads on different two sides by using a magnetic rack, fully washing the magnetic beads, adsorbing for 2min by using the magnetic rack, removing the supernatant, and standing at room temperature until the ethanol volatilizes cleanly;

9. The method for detecting a mixed sample according to claim 2, wherein,

In step 9, the two-round PCR procedure: 3min at 95 ℃; 15s at 95 ℃, 15s at 58 ℃, 30s at 72 ℃ and 12 cycles; and at 72℃for 4min.

10. The method for detecting a mixed sample according to claim 2, wherein,

In step 10, the two rounds of PCR products were purified using 0.80-fold beads, as follows:

Step 10.4, adding 23 μl of an adsorption Buffer, fully suspending magnetic beads, standing at room temperature for 2min to elute DNA, adsorbing the magnetic beads with a magnet, and sucking the obtained supernatant DNA solution into a new tube to obtain a sequencing library; the said solution Buffer is 10mM Tris-HCl, pH 8.0-8.5.