CN113969322B - SNP core site, primer and high-throughput purity identification method for identifying purity of corn hybrid - Google Patents
SNP core site, primer and high-throughput purity identification method for identifying purity of corn hybrid Download PDFInfo
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Abstract
The application provides a set of SNP core sites for identifying the purity of corn hybrid seeds, primers and a method for identifying the purity in high throughput, belonging to the technical field of genetic engineering, wherein the SNP core sites comprise 20 core sites such as 8974336 th site of corn chromosome 1, 289408285 th site of chromosome 1 and the like. The SNP locus provided by the application can be used for identifying the purity of corn hybrid.
Description
Technical Field
The application belongs to the technical field of genetic engineering, and particularly relates to a set of SNP core loci and primers for identifying the purity of corn hybrid seeds and a method for identifying the purity.
Background
The purity of the corn hybrid is one of the key factors affecting the field yield, the product quality and the income of farmers. Therefore, the corn hybrid purity identification is an important means for controlling the quality of corn seeds, and particularly provides important basis for detecting the selfed seedlings in the hybrid, and providing a plurality of links such as quality automatic control of seed enterprises, government market monitoring and the like.
The current method for identifying the purity of the corn hybrid seeds mainly comprises a field cell planting identification method, a salt-soluble protein method, an SSR molecular marker method and the like. Field cell planting identification is a purity identification method widely used at present, and is also marked as the most accurate identification method in the inspection procedure, but the method has some problems, mainly comprising the following aspects. First, the identification period is long, and the result can be obtained only by waiting for one planting period, so that the purity problem can not be found in time and the economic loss can be reduced to the minimum. Secondly, the result is easily influenced by environmental factors, and the weak plants and the disease plants generated by uneven ground force or insect diseases are easily mixed with the selfing plants, so that erroneous judgment or missed judgment results appear, and the purity results are finally influenced. Because the planting ecological areas suitable for the corn varieties are different, if the planting identification is not in the ecological areas suitable for the varieties, the varieties have large phenotype change, and the difficulty of judgment is increased. Thirdly, the requirements on the inspectors are high, the inspectors are easily influenced by personnel factors, the inspectors need to fully know the characteristics of different varieties and the differences between the characteristics and the parent or similar varieties, and the inspectors are required to have rich field identification experience.
The method for identifying the purity of the maize varieties is also widely applied to the purity identification of the maize varieties due to the rapidness, simplicity and convenience, but the method also has some problems that the protein electrophoresis cannot find the parental difference on certain varieties or cannot distinguish the selfing seedlings, and cannot identify all varieties. The SSR and other molecular marker identification methods cannot meet the high-throughput detection requirement of samples, and particularly meet the requirement of short-term mass variety purity identification during the period from seed production to marketing.
For hybrid purity identification, the requirement of short-term mass variety purity identification from the seed production link to the marketing period is met, so that the exploration of a set of high-throughput schemes which are efficient, accurate and suitable for the purity identification of most corn hybrid varieties in the market is of great significance.
Disclosure of Invention
Accordingly, the present application aims to provide a set of SNP core sites, primers and a method for high throughput purity identification for identifying the purity of corn hybrid seeds.
In order to achieve the above object, the present application provides the following technical solutions:
the application provides a set of SNP core sites for identifying the purity of corn hybrid, which comprises a 8974336 th bit of corn chromosome 1, a 289408285 th bit of chromosome 1, a 109563976 th bit of chromosome 2, a 186339948 th bit of chromosome 2, a 27775731 th bit of chromosome 3, a 118083714 th bit of chromosome 3, a 20077010 th bit of chromosome 4, a 128874466 th bit of chromosome 4, a 13402375 th bit of chromosome 5, a 204879476 th bit of chromosome 5, a 39822979 th bit of chromosome 6, a 141476300 th bit of chromosome 6, a 126677146 th bit of chromosome 7, a 15521714 th bit of chromosome 7, a 20978845 th bit of chromosome 8, a 118299376 th bit of chromosome 9, a 104695670 th bit of chromosome 9, a 127197714 th bit of chromosome 9, a 39960289 th bit of chromosome 10 and a 109396730 th bit of chromosome 10.
The application also provides a group of primers for amplifying the SNP core locus according to the technical scheme, and the nucleotide sequence of the primers is shown as SEQ ID No. 1-60.
Preferably, the nucleotide sequence of 8974336 th bit of the amplified corn chromosome 1 is shown in SEQ ID No. 1-3;
the nucleotide sequence of 289408285 th bit of the amplified corn chromosome 1 is shown in SEQ ID No. 4-6;
the nucleotide sequence of 109563976 th bit of the amplified corn chromosome 2 is shown in SEQ ID No. 7-9;
the nucleotide sequence of 186339948 th bit of the amplified corn chromosome 2 is shown in SEQ ID No. 10-12;
the nucleotide sequence of 27775731 th bit of the amplified corn chromosome 3 is shown in SEQ ID No. 13-15;
the nucleotide sequence of 118083714 th bit of the amplified corn chromosome 3 is shown in SEQ ID No. 16-18;
the nucleotide sequence of 20077010 th bit of the amplified corn chromosome 4 is shown in SEQ ID No. 19-21;
the nucleotide sequence of 128874466 th bit of the amplified corn chromosome 4 is shown in SEQ ID No. 22-24;
the nucleotide sequence of 13402375 th bit of the amplified corn chromosome 5 is shown in SEQ ID No. 25-27;
the nucleotide sequence of 204879476 th bit of the amplified corn chromosome 5 is shown in SEQ ID No. 28-30;
the nucleotide sequence of 39822979 th bit of the amplified corn chromosome 6 is shown in SEQ ID No. 31-33;
the nucleotide sequence of 141476300 th bit of the amplified corn chromosome 6 is shown in SEQ ID No. 34-36;
the nucleotide sequence of 126677146 th bit of the amplified corn chromosome 7 is shown in SEQ ID No. 37-39;
the nucleotide sequence of 15521714 th bit of the amplified corn chromosome 7 is shown in SEQ ID No. 40-42;
the nucleotide sequence of 20978845 th bit of the amplified corn chromosome 8 is shown as SEQ ID No. 43-45;
the nucleotide sequence of 118299376 th bit of the amplified corn chromosome 8 is shown in SEQ ID No. 46-48;
the nucleotide sequence of 104695670 th bit of the amplified corn chromosome 9 is shown in SEQ ID No. 49-51;
the nucleotide sequence of 127197714 th bit of the amplified corn chromosome 9 is shown as SEQ ID No. 52-54;
the nucleotide sequence of 39960289 th bit of the amplified corn chromosome 10 is shown as SEQ ID No. 55-57;
the nucleotide sequence of 109396730 th bit of the amplified corn chromosome 10 is shown in SEQ ID No. 58-60.
The application also provides a method for identifying the purity of the corn hybrid by using the primer in high flux, which comprises the following steps:
1) Extracting DNA of corn materials, and carrying out PCR on the DNA by using the primer in the technical scheme;
2) And (3) collecting an original fluorescent signal, then carrying out genotyping, introducing the obtained genotyping result into a SNP database management system, removing the sites of non-complementation and genetic instability of parents, counting the results of at least two sites, and calculating the purity of the corn.
Preferably, the step 1) corn comprises kernels, seedlings or leaves of corn.
Preferably, the system of the PCR of the step 1) comprises per 1 μl: 20ng of dried DNA, 0.5. Mu.L of 2 XKASP PCR premix, 0.486. Mu.L of deionized water and 0.014. Mu.L of primer working solution.
Preferably, the system of the PCR of the step 1) comprises per 3. Mu.L: 1.45. Mu.L of DNA, 1.5. Mu.L of 2 XKASP PCR premix and 0.05. Mu.L of primer working solution.
Preferably, the system of the PCR of the step 1) comprises per 10. Mu.L: 1.5. Mu.L of DNA, 5. Mu.L of 2 XKASP PCR premix, 3.36. Mu.L of deionized water and 0.14. Mu.L of primer working solution.
Preferably, the concentration of the upstream primer in the primer working solution is 12 mu mol/L, and the concentration of the downstream primer is 30 mu mol/L.
Preferably, the PCR procedure comprises: 94 ℃ for 15min;94℃for 20s,61-55℃for 1min for 10 cycles; 94℃for 20s,55℃for 60s,30 cycles.
The application provides a set of SNP core sites for identifying the purity of corn hybrid, which comprises a 8974336 th bit of corn chromosome 1, a 289408285 th bit of chromosome 1, a 109563976 th bit of chromosome 2, a 186339948 th bit of chromosome 2, a 27775731 th bit of chromosome 3, a 118083714 th bit of chromosome 3, a 20077010 th bit of chromosome 4, a 128874466 th bit of chromosome 4, a 13402375 th bit of chromosome 5, a 204879476 th bit of chromosome 5, a 39822979 th bit of chromosome 6, a 141476300 th bit of chromosome 6, a 126677146 th bit of chromosome 7, a 15521714 th bit of chromosome 7, a 20978845 th bit of chromosome 8, a 118299376 th bit of chromosome 9, a 104695670 th bit of chromosome 9, a 127197714 th bit of chromosome 9, a 39960289 th bit of chromosome 10 and a 109396730 th bit of chromosome 10. The locus provided by the application can be used for fixing the purity of corn hybrid seeds.
Drawings
FIG. 1 is a graph of test site typing effect (MG 279), a is CTAB typing effect, and b is quick-extraction typing effect;
FIG. 2 is a graph showing the comparison of the sample parental complementary genotype ratios, with the abscissa showing the ranking of samples from low to high according to the 20-site parental complementary ratios, and the ordinate showing the sample parental complementary genotype ratios;
FIG. 3 is a statistical plot of the ratio intervals of the parental complementary genotypes of the samples, and the histogram shows the number of samples per 10% increase in heterozygosity.
Detailed Description
The application provides a set of SNP core sites for identifying the purity of corn hybrid, which comprises a 8974336 th bit of corn chromosome 1, a 289408285 th bit of chromosome 1, a 109563976 th bit of chromosome 2, a 186339948 th bit of chromosome 2, a 27775731 th bit of chromosome 3, a 118083714 th bit of chromosome 3, a 20077010 th bit of chromosome 4, a 128874466 th bit of chromosome 4, a 13402375 th bit of chromosome 5, a 204879476 th bit of chromosome 5, a 39822979 th bit of chromosome 6, a 141476300 th bit of chromosome 6, a 126677146 th bit of chromosome 7, a 15521714 th bit of chromosome 7, a 20978845 th bit of chromosome 8, a 118299376 th bit of chromosome 9, a 104695670 th bit of chromosome 9, a 127197714 th bit of chromosome 9, a 39960289 th bit of chromosome 10 and a 109396730 th bit of chromosome 10.
The application also provides a group of primers for amplifying the SNP core locus according to the technical scheme, and the nucleotide sequence of the primers is shown as SEQ ID No. 1-60.
In the application, the 8974336 nucleotide sequence of the amplified corn chromosome 1 is shown in SEQ ID No. 1-3; the nucleotide sequence of 289408285 th bit of the amplified corn chromosome 1 is shown in SEQ ID No. 4-6; the nucleotide sequence of 109563976 th bit of the amplified corn chromosome 2 is shown in SEQ ID No. 7-9; the nucleotide sequence of 186339948 th bit of the amplified corn chromosome 2 is shown in SEQ ID No. 10-12; the nucleotide sequence of 27775731 th bit of the amplified corn chromosome 3 is shown in SEQ ID No. 13-15; the nucleotide sequence of 118083714 th bit of the amplified corn chromosome 3 is shown in SEQ ID No. 16-18; the nucleotide sequence of 20077010 th bit of the amplified corn chromosome 4 is shown in SEQ ID No. 19-21; the nucleotide sequence of 128874466 th bit of the amplified corn chromosome 4 is shown in SEQ ID No. 22-24; the nucleotide sequence of 13402375 th bit of the amplified corn chromosome 5 is shown in SEQ ID No. 25-27; the nucleotide sequence of 204879476 th bit of the amplified corn chromosome 5 is shown in SEQ ID No. 28-30; the nucleotide sequence of 39822979 th bit of the amplified corn chromosome 6 is shown in SEQ ID No. 31-33; the nucleotide sequence of 141476300 th bit of the amplified corn chromosome 6 is shown in SEQ ID No. 34-36; the nucleotide sequence of 126677146 th bit of the amplified corn chromosome 7 is shown in SEQ ID No. 37-39; the nucleotide sequence of 15521714 th bit of the amplified corn chromosome 7 is shown in SEQ ID No. 40-42; the nucleotide sequence of 20978845 th bit of the amplified corn chromosome 8 is shown as SEQ ID No. 43-45; the nucleotide sequence of 118299376 th bit of the amplified corn chromosome 8 is shown in SEQ ID No. 46-48; the nucleotide sequence of 104695670 th bit of the amplified corn chromosome 9 is shown in SEQ ID No. 49-51; the nucleotide sequence of 127197714 th bit of the amplified corn chromosome 9 is shown as SEQ ID No. 52-54; the nucleotide sequence of 39960289 th bit of the amplified corn chromosome 10 is shown as SEQ ID No. 55-57; the nucleotide sequence of 109396730 th bit of the amplified corn chromosome 10 is shown in SEQ ID No. 58-60.
In the present application, the nucleotide sequences of the primers are specifically shown in Table 1.
TABLE 1 identification of corn purity SNP core site table and primer sequences
In the application, the 5' end of the forward primer 1 is preferably connected with a FAM fluorescence corresponding adaptor sequence; and HEX fluorescence corresponding linker sequence is connected to the 5' end of the forward primer 2.
The application also provides a method for identifying the purity of the corn hybrid by using the primer in high flux, which comprises the following steps:
1) Extracting DNA of corn materials, and carrying out PCR on the DNA by using the primer in the technical scheme;
2) And (3) collecting an original fluorescent signal, then carrying out genotyping, introducing the obtained genotyping result into a SNP database management system, removing the sites of non-complementation and genetic instability of parents, counting the results of at least two sites, and calculating the purity of the corn.
In the present application, the corn preferably comprises kernels, seedlings or leaves of corn. The method for extracting the corn DNA is not particularly limited, and a conventional method for extracting plant DNA is adopted, and the method for extracting the corn DNA is particularly preferably adopted in the specific embodiment of the application.
In the present application, the system for PCR preferably comprises, per 1. Mu.L: 20ng of dried DNA, 0.5. Mu.L of 2 XKASP PCR premix, 0.486. Mu.L of deionized water and 0.014. Mu.L of primer working solution. In the present application, the system of PCR preferably comprises, per 3. Mu.L: 1.45. Mu.L of DNA, 1.5. Mu.L of 2 XKASP PCR premix and 0.05. Mu.L of primer working solution. In the present application, the system for PCR preferably comprises, per 10. Mu.L: 1.5. Mu.L of 2 XKASP PCR premix, 3.36. Mu.L of deionized water and 0.14. Mu.L of primer working solution. In the present application, the concentration of the upstream primer in the primer working solution is preferably 12. Mu. Mol/L, and the concentration of the downstream primer is preferably 30. Mu. Mol/L. In the present application, the PCR process preferably includes: 94 ℃ for 15min;94℃for 20s,61-55℃for 1min for 10 cycles; 94℃for 20s,55℃for 60s,30 cycles.
The present application preferably uses a Pherastar fluorescence scanner to collect the raw fluorescence signal. Genotyping is preferably performed using the Kraken (v16.3.16.16288) software. The present application preferably directs the typing results to a SNP database management system (V1.0.0, software accession number 2018SR 043088) for analysis of corn purity.
The technical solutions provided by the present application are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present application.
Example 1
Determination of core primers for purity identification of maize hybrid
1. Experimental samples: test materials 12 sets of triplet samples (table 2).
Table 2 corn triplet sample information table
| Numbering device | Hybrid seeds | Female parent | Father parent |
| 1 | Jade 335 | PH6WC | PH4CV |
| 2 | Zhengdan 958 | Zheng 58 | Chang 7-2 |
| 3 | Nongda 108 | X178 | Yellow C |
| 4 | Ruuzuan 981 | Zizang 319 | Lx9801 |
| 5 | Shen Shan 16 and Shen Shan | K12 | Shen 137 |
| 6 | Dongdan 60 | A801 | Dan 598 |
| 7 | Danyu 39 | C8605-2 | Dan 598 |
| 8 | Zhongdan No.2 | Mo17 | Self 330 |
| 9 | Tuck sheet 13 | Tuck 478 | Dan 340 |
| 10 | SC704 | B73 | Mo17 |
| 11 | Jingke glutinous 2000 | Jing glutinous rice 6 | White glutinous rice 6 |
| 12 | Nonghua 101 | NH60 | S121 |
2. Nucleic acid extraction: the triplet sample is extracted by adopting a modified CTAB method mixed strain. The anonymous hybrid sample is extracted by an improved DNA quick extraction method (K-MateDNA extraction kit, LGC genome), 100 mu L of 1-x extracting solution is added into a micro-pore plate, the micro-pore plate is placed at 95 ℃ for 15min and 4 ℃ for 10min, 100 mu L of 1-x buffer solution is added, the mixture is evenly mixed at 1000rpm, centrifugation is carried out at 2500rpm for 30s, standing is carried out, a proper amount of supernatant is absorbed, and 5 times of deionized water is added to prepare working solution for standby.
3. Purity candidate site and primer design: according to the parameters of SNP fingerprint data of 384SNP loci of 335 national standard trial corn hybrid samples, such as heterozygosity rate, minimum allele frequency (MAF, minor Allele Frequency), polymorphism information content (PIC, polymorphic Information Content) and the like of each locus, 60 loci with uniform distribution on chromosomes and good polymorphism are selected as purity candidate loci (table 3). Designing and synthesizing KASP primers from 60 candidate sites, adding a joint sequence of FAM or HEX fluorescent groups at the 5' ends of the two forward primer sequences, and mixing the forward primer with the downstream primer according to 12 mu mol/L of the upstream primer and 30 mu mol/L of the downstream primer to prepare primer working solution for later use.
TABLE 3 information table of SNP candidate site for identifying corn purity
PCR reaction system: 20ng of dried DNA, 0.5. Mu.L of 2 XKASP PCR premix (LGC Genomic,1536 format, standard content ROX, UK), 0.486. Mu.L of deionized water, 0.014. Mu.L of primer working solution. The reaction procedure: 94 ℃ for 15min;94℃for 20s,61-55℃for 1min for 10 cycles; 94℃for 20s,55℃for 60s,30 cycles.
5. Fluorescence, data acquisition and analysis: the original fluorescence signal was collected using a Pherastar fluorescence scanner, genotyping was performed using Kraken (v16.3.16.16288) software, and the results of the genotyping were imported into the SNP database management system (V1.0.0, software accession number: 2018SR 043088) for analysis of sample purity. The parameters of site heterozygosity, MAF, PIC, etc. were calculated using SNP alignment statistical tool software (V1.0, software accession number: 2018SR 026743).
6. Test results: based on the corn 384SNP basic locus combination, 1 set of core locus combination suitable for corn hybrid purity identification is screened and determined according to conditions such as locus parent-parent complementation distinguishing capability, test parting effect, uniform dyeing distribution and the like. The specific screening process is as follows: (1) determining candidate site combinations: based on fingerprint data of 335 basic sites of national audit varieties, parameters such as site parent complementarity rate, polymorphism and the like are calculated, the screening condition is that the site parent complementarity rate is higher than 40%, MAF is more than or equal to 0.3, PIC is more than or equal to 0.3, and 60 candidate sites are optimized. (2) KASP primer evaluation: candidate sites were designed to synthesize KASP primers and tested using 12 sets of maize hybrids and their parental triplet materials. Wherein 57 primers can be divided into three groups closely, the typing results are consistent with the expected results, the Mendelian's genetic law is met, and the test repetition results are consistent (a in FIG. 1). (3) rapid DNA extraction assessment: to shorten the DNA preparation time and improve the efficiency of purity identification, the rapid extraction method was further used to test sites that perform well in CTAB typing, and a total of 48 sites were clearly separated into three closely typed sites (b in fig. 1) that were consistent with the expected typing results using both CTAB and rapid extraction methods. (4) determining the purity to identify the core site: 20 sites were selected as the purity-identified core sites by integrating the site discrimination ability and the chromosome uniform distribution (Table 1).
Example 2
SNP core site discrimination capability verification
1. Experimental samples: the test material is 335 parts of national approval corn variety fingerprint data (list is omitted) in 1984-2013.
2. Nucleic acid extraction: at least one blank was placed per 95 samples extracted using the modified CTAB method (same method as example 1).
3. Primer, reaction system and reaction procedure: as in example 1.
4. Fluorescence, data acquisition and analysis: as in example 1.
5. Test results: the sample parental complementary genotype ratio was 83.6% higher at the core site than at the base site (fig. 2). Of 335 hybrid samples, 99.7% of the samples had a ratio of more than 10% of the parental complementary genotypes, and multiple parental complementary sites were available for screening for purity identification (FIG. 3). According to the comprehensive parameter description, 20 purity identification core sites are obviously superior to basic sites and candidate sites, and the 20 core site combinations can be effectively applied to purity identification of most corn hybrids in the current domestic market.
Example 3
Purity identification of samples for testing using corn purity core site
1. Experimental samples: several samples of genitals 968 seeds were tested, and genitals 968 parent seeds.
2. Nucleic acid extraction: the DNA working solution is prepared for standby by adopting an improved DNA quick extraction method (K-Mate DNA extraction kit, LGC Genomic) single plant extraction. At least 2 parental DNA and one blank were placed per 93 individuals.
3. Primer, reaction system and reaction procedure: as in example 1.
4. Fluorescence, data acquisition and analysis: as in example 1.
5. Removing the sites of non-complementation and genetic instability of the parents, counting the results of at least two sites, and calculating the purity of the sample.
6. Test results: the sample was shown to be genotyped as a parental complement at 15 out of 20 core sites, from which four sites MG072, MG135, MG175, MG215 were selected for purity identification. Among 110 individuals tested, 1 selfed strain and 2 abnormal strains were detected together, and the purity of the sample was 97.3%. According to national standard, the purity of the corn single cross is not lower than 96.0%, and the experimental result shows that the purity of the sample meets the standard. The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.
Sequence listing
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<213> Artificial sequence (Artificial Sequence)
<400> 23
ctagcttagc agagctgcat ctt 23
<210> 24
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
cccacgtcac ctagataagc caaaa 25
<210> 25
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
gatatttctg caactaaaca tggcaag 27
<210> 26
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
ggatatttct gcaactaaac atggcaaa 28
<210> 27
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
atactggggt tgtggggata ggatt 25
<210> 28
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
acttctccat cctcttccaa catatta 27
<210> 29
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
cttctccatc ctcttccaac atattg 26
<210> 30
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
agctgtccac catcagtact ggaat 25
<210> 31
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
agatggcatt gtgatctgtg caca 24
<210> 32
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
gatggcattg tgatctgtgc acg 23
<210> 33
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
agccgaagga ttgatcctcc tcat 24
<210> 34
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
gaaacatgaa tgccctaaat ccttcg 26
<210> 35
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
agaaacatga atgccctaaa tccttct 27
<210> 36
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
attatgttca ccaagtatcc agatggcat 29
<210> 37
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
ggttccatgg ctacctgaca agt 23
<210> 38
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
gttccatggc tacctgacaa gg 22
<210> 39
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
taggagctag ccaagagcct acta 24
<210> 40
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
aacgtatgag atgaactcac cagaaa 26
<210> 41
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
acgtatgaga tgaactcacc agaag 25
<210> 42
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
ctccgccgct ggtggagcta 20
<210> 43
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
gacctgaaat gcttggcgag tca 23
<210> 44
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
acctgaaatg cttggcgagt cc 22
<210> 45
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
gcaggagcct tagcgtggct at 22
<210> 46
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
cctcagatct catctatgct gcc 23
<210> 47
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
cctcagatct catctatgct gct 23
<210> 48
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
cgtttccaca ttttctgaag gtttcacaa 29
<210> 49
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
aacacgagct ggttgatgga ttagt 25
<210> 50
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
cacgagctgg ttgatggatt agc 23
<210> 51
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
gcctctggta cgttagtttg cagtt 25
<210> 52
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
tactgaccga gcgatgctgc t 21
<210> 53
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
ctgaccgagc gatgctgcc 19
<210> 54
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
cgctgatggt cacagaaaca tcgtt 25
<210> 55
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
gaagcaatcc ttccggagga atg 23
<210> 56
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
gaagcaatcc ttccggagga ata 23
<210> 57
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
gaatgtgcag attggatttg agggataaa 29
<210> 58
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
ggttgacatg agacttgcag aga 23
<210> 59
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
ggttgacatg agacttgcag agg 23
<210> 60
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
tcgggaagcc atacttcaca tgcat 25
<210> 61
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 61
ccggagctgg cttctgaatc aa 22
<210> 62
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
cggagctggc ttctgaatca g 21
<210> 63
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
acatccccat cctggggagg aa 22
<210> 64
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
gagcgtcaac gacaaagcca agt 23
<210> 65
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 65
agcgtcaacg acaaagccaa gc 22
<210> 66
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 66
cctgttaggt tgtaagaatt gagccttat 29
<210> 67
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 67
ggagatgctc tacaagtttg tca 23
<210> 68
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 68
ggagatgctc tacaagtttg tcg 23
<210> 69
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 69
ccttcgagga ggccaaggac tt 22
<210> 70
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 70
ccatggtttt aaggaactat cgaaaga 27
<210> 71
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 71
catggtttta aggaactatc gaaagg 26
<210> 72
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 72
atcaatgtac tcccataagc agcaacttt 29
<210> 73
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 73
catcgaatgg ctgatcatgt tgcat 25
<210> 74
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 74
atcgaatggc tgatcatgtt gcac 24
<210> 75
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 75
ccattccctg tatgacagac acgat 25
<210> 76
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 76
gcccctgcat tgtttgcagc 20
<210> 77
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 77
ctgcccctgc attgtttgca gt 22
<210> 78
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 78
gccactgcaa atccaaagaa tccgta 26
<210> 79
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 79
aggatcacaa tccatctgct gcaaa 25
<210> 80
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 80
gatcacaatc catctgctgc aac 23
<210> 81
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 81
cctgcagttg ctactgatag ttctcaa 27
<210> 82
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 82
atgtttctca ggacggtaat agtgat 26
<210> 83
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 83
gtttctcagg acggtaatag tgac 24
<210> 84
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 84
cccatccatt ccacatattc ggcaa 25
<210> 85
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 85
cggcctccat gcttgatgat g 21
<210> 86
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 86
ccggcctcca tgcttgatga ta 22
<210> 87
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 87
gtcggtcgag tcaaaattca ttttggat 28
<210> 88
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 88
attcgaactg ctgccttgac taatc 25
<210> 89
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 89
attcgaactg ctgccttgac taata 25
<210> 90
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 90
gcagcagtga ctatccttct gaagaa 26
<210> 91
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 91
ccaatcaagg cggcaacata cc 22
<210> 92
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 92
accaatcaag gcggcaacat act 23
<210> 93
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 93
gcgttcatgt tcatggaagg ccaaa 25
<210> 94
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 94
attctgaatg taaaacttaa catgctgcta 30
<210> 95
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 95
ctgaatgtaa aacttaacat gctgctg 27
<210> 96
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 96
aagtccttcc aactttcagc ataagcaaa 29
<210> 97
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 97
ggttacacga ccaaatgagt accat 25
<210> 98
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 98
gttacacgac caaatgagta ccag 24
<210> 99
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 99
taggcagagc agccattgac aagta 25
<210> 100
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 100
tatcacttgt ggatctatat ctgtg 25
<210> 101
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 101
gcttatcact tgtggatcta tatctgtt 28
<210> 102
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 102
tgaacccaaa gcctcggtgt tcttt 25
<210> 103
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 103
atacagtgaa acagcttgca ctgga 25
<210> 104
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 104
cagtgaaaca gcttgcactg gg 22
<210> 105
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 105
ttaatttttg gaagagcttg cgttgggaa 29
<210> 106
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 106
agttacctgt catcgatctc tggat 25
<210> 107
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 107
acctgtcatc gatctctgga c 21
<210> 108
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 108
aaagccctct gacaatgctc cagta 25
<210> 109
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 109
cctctgtaag cgcagtactg gt 22
<210> 110
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 110
ctctgtaagc gcagtactgg c 21
<210> 111
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 111
aaattgctat gcaaacaggt tctggagta 29
<210> 112
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 112
gagctagtaa atattgttgt tgttcctc 28
<210> 113
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 113
agagctagta aatattgttg ttgttcctt 29
<210> 114
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 114
cgccgacggg acgacggat 19
<210> 115
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 115
cataaacagt aggtttatcg ctgacataa 29
<210> 116
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 116
aaacagtagg tttatcgctg acatag 26
<210> 117
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 117
gtgataaccg atgcaaaatg ctgcttaat 29
<210> 118
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 118
ccaaaggata gcacatcttg gtg 23
<210> 119
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 119
gtccaaagga tagcacatct tggta 25
<210> 120
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 120
tgtcaaccgc atcctggcag ataat 25
<210> 121
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 121
gacgacgact ccatcgtgac c 21
<210> 122
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 122
gacgacgact ccatcgtgac a 21
<210> 123
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 123
tcaacccatg gctgctcaca tgtaa 25
<210> 124
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 124
ggcattctga tttgacagcc cac 23
<210> 125
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 125
ggcattctga tttgacagcc caa 23
<210> 126
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 126
tcctgattct gtacttgatt ggaccaaa 28
<210> 127
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 127
gcagctgaga aacaattgca aagtg 25
<210> 128
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 128
gcagctgaga aacaattgca aagta 25
<210> 129
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 129
gtactctcag atggttttgt gacatcaa 28
<210> 130
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 130
gtgctcgaac gaatcgacca g 21
<210> 131
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 131
cgtgctcgaa cgaatcgacc aa 22
<210> 132
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 132
catccatggc gaagctcatg aacaa 25
<210> 133
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 133
gtagcgtgtc tctacgctct g 21
<210> 134
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 134
atgtagcgtg tctctacgct ctt 23
<210> 135
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 135
cagcgcgtta cgacgaactc caa 23
<210> 136
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 136
cagcgacctc aagaagttga agtaa 25
<210> 137
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 137
agcgacctca agaagttgaa gtag 24
<210> 138
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 138
gtacgacatg cagtttgaca tcaagtat 28
<210> 139
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 139
gaagctacta ttagcaatga tctatatgat 30
<210> 140
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 140
aagctactat tagcaatgat ctatatgac 29
<210> 141
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 141
acaggattga taaacattac ctgcaggaa 29
<210> 142
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 142
gatcgttgtc ttcacaaatg aagaatagt 29
<210> 143
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 143
cgttgtcttc acaaatgaag aatagc 26
<210> 144
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 144
gcgagatatt gaaagctagt ggtgcta 27
<210> 145
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 145
gaactaactg agtgttaaag gagcttat 28
<210> 146
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 146
aactaactga gtgttaaagg agcttag 27
<210> 147
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 147
ccttgacaca accgctctcc ttaaa 25
<210> 148
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 148
aaccattccc ttcatacttc ttctct 26
<210> 149
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 149
accattccct tcatacttct tctcc 25
<210> 150
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 150
gggagtatct tttaggaaga tgtacagat 29
<210> 151
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 151
caatccaaag cagaaagaag ttgttct 27
<210> 152
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 152
aatccaaagc agaaagaagt tgttcc 26
<210> 153
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 153
ccaaaacagt gaagtgaccg ccat 24
<210> 154
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 154
caaagtggtg taaatggatg gatcg 25
<210> 155
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 155
caaagtggtg taaatggatg gatca 25
<210> 156
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 156
ttggacactc caggggatcc tata 24
<210> 157
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 157
gcctcacaca tccatatacg tagaa 25
<210> 158
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 158
cctcacacat ccatatacgt agag 24
<210> 159
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 159
cttccatgca tcgccctatg gatat 25
<210> 160
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 160
aacactcatg tctgctccag gg 22
<210> 161
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 161
aaacactcat gtctgctcca gga 23
<210> 162
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 162
tcgatgtttt cgatcccaag ttcaacatt 29
<210> 163
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 163
ccacttctgc tcgtatgatc ttc 23
<210> 164
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 164
gccacttctg ctcgtatgat ctta 24
<210> 165
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 165
tcccgtaatc atctgctcgt ctgta 25
<210> 166
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 166
ataccctctc caccagttgt tgat 24
<210> 167
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 167
accctctcca ccagttgttg ac 22
<210> 168
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 168
tcgcagggag gcgtcgttca a 21
<210> 169
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 169
aaaagtgcag ttccttgctg ttcattt 27
<210> 170
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 170
aagtgcagtt ccttgctgtt cattg 25
<210> 171
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 171
cccaatgagc aaaaagaata gcaccaaa 28
<210> 172
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 172
tgttccgaat agcaagtgat ctcttt 26
<210> 173
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 173
gttccgaata gcaagtgatc tcttc 25
<210> 174
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 174
gggaaacctg cagaatgctg ttgat 25
<210> 175
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 175
caagtgcgca gcaagccaaa ag 22
<210> 176
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 176
aaacaagtgc gcagcaagcc aaaat 25
<210> 177
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 177
ccgttcttaa gcgctccatc ctttt 25
<210> 178
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 178
cacgaagctc tcgcgctctt c 21
<210> 179
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 179
cacgaagctc tcgcgctctt t 21
<210> 180
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 180
ggcatggagc ccctatcctt gat 23
Claims (8)
1. A group of primer groups for amplifying SNP core sites with corn hybrid purity, which is characterized in that the primer groups consist of primers shown as SEQ ID No. 1-60,
the nucleotide sequence of 8974336 th bit of the amplified corn chromosome 1 is shown as SEQ ID No. 1-3;
the 289408285 nucleotide sequence of the amplified corn chromosome 1 is shown in SEQ ID No. 4-6;
the 109563976 nucleotide sequence of the amplified corn chromosome 2 is shown in SEQ ID No. 7-9;
the 186339948 nucleotide sequence of the amplified corn chromosome 2 is shown in SEQ ID No. 10-12;
the 27775731 nucleotide sequence of the amplified corn chromosome 3 is shown in SEQ ID No. 13-15;
the 118083714 nucleotide sequence of the amplified corn chromosome 3 is shown as SEQ ID No. 16-18;
the 20077010 nucleotide sequence of the amplified corn chromosome 4 is shown in SEQ ID No. 19-21;
the 128874466 nucleotide sequence of the amplified corn chromosome 4 is shown in SEQ ID No. 22-24;
the 13402375 nucleotide sequence of the amplified corn chromosome 5 is shown in SEQ ID No. 25-27;
the 204879476 nucleotide sequence of the amplified corn chromosome 5 is shown in SEQ ID No. 28-30;
the 39822979 nucleotide sequence of the amplified corn chromosome 6 is shown in SEQ ID No. 31-33;
the 141476300 nucleotide sequence of the amplified corn chromosome 6 is shown as SEQ ID No. 34-36;
the nucleotide sequence of 126677146 th bit of the amplified corn chromosome 7 is shown as SEQ ID No. 37-39;
the 15521714 nucleotide sequence of the amplified corn chromosome 7 is shown as SEQ ID No. 40-42;
the 20978845 nucleotide sequence of the amplified corn chromosome 8 is shown as SEQ ID No. 43-45;
the 118299376 nucleotide sequence of the amplified corn chromosome 8 is shown as SEQ ID No. 46-48;
the 104695670 nucleotide sequence of the amplified corn chromosome 9 is shown in SEQ ID No. 49-51;
the 127197714 nucleotide sequence of the amplified corn chromosome 9 is shown as SEQ ID No. 52-54;
the 39960289 nucleotide sequence of the amplified corn chromosome 10 is shown as SEQ ID No. 55-57;
the nucleotide sequence of 109396730 th bit of the amplified corn chromosome 10 is shown as SEQ ID No. 58-60.
2. A method for high throughput identification of maize hybrid purity using the primer set of claim 1, comprising the steps of:
1) Extracting DNA of corn material, and performing PCR on the DNA by using the primer set of claim 1;
2) And (3) collecting an original fluorescent signal, then carrying out genotyping, introducing the obtained genotyping result into a SNP database management system, removing the sites of non-complementation and genetic instability of parents, counting the results of at least two sites, and calculating the purity of the corn.
3. The method of claim 2, wherein the step 1) corn material comprises kernels, seedlings or leaves of corn.
4. The method according to claim 2, wherein the system of step 1) PCR comprises per 1 μl: 20ng of dried DNA, 0.5. Mu.L of 2 XKASP PCR premix, 0.486. Mu.L of deionized water and 0.014. Mu.L of primer working solution.
5. The method according to claim 2, wherein the system of step 1) PCR comprises per 3 μl: 1.45. Mu.L of DNA, 1.5. Mu.L of 2 XKASP PCR premix and 0.05. Mu.L of primer working solution.
6. The method according to claim 2, wherein the system of step 1) PCR comprises per 10 μl: 1.5. Mu.L of DNA, 5. Mu.L of 2 XKASP PCR premix, 3.36. Mu.L of deionized water and 0.14. Mu.L of primer working solution.
7. The method according to any one of claims 4 to 6, wherein the concentration of the upstream primer in the primer working solution is 12. Mu. Mol/L and the concentration of the downstream primer is 30. Mu. Mol/L.
8. The method of any one of claims 2, 4-6, wherein the PCR procedure comprises: 94. 15min at the temperature; 94. 20s at the temperature of 61-55 ℃ for 1min for 10 cycles; 94. 20s at C, 60s at 55℃,30 cycles.
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