Corn triallelic variation SNP site combination and application thereof

Abstract

The invention relates to the technical field of molecular identification of crops, in particular to a corn triallelic variation SNP site combination and application thereof. The triple allelic variation SNP site combination comprises 40 triple allelic variation SNP sites, and the triple allelic variation SNP site combination comprises nucleotide sequences as shown in SEQ ID NO.1-40. The SNP site combination is obtained through screening and can be applied to corn variety authenticity identification, genetic relationship identification, purity identification and plant breeding. Compared with diallelic variation sites, the 40 triallelic variation SNP sites provided by the invention have the advantages that the variety recognition capability of a single site is greatly improved, and the development and application of the 40 triallelic variation SNP sites expand the range of available marker sites of corn on the genome level; the invention provides a new thought and method for corn variety identification, genetic relationship evaluation and the like, and has important value in the field of corn breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular identification of crops, and particularly to a combination of maize tri-allelic variant SNP loci and its application. Background Art

[0002] The rapid development of high-throughput sequencing, bioinformatics, and genotyping technologies has made it possible to effectively identify crop varieties using newer and more efficient molecular techniques. Recently, the molecular identification technology of crop varieties based on SSR markers has been widely used in the seed industry. However, with the continuous change of detection requirements, the SSR marker technology has been difficult to meet the higher requirements for the number of markers, detection throughput, fingerprint integration and sharing, etc.

[0003] SNP is a marker locus developed based on single nucleotide polymorphism, which has characteristics such as high distribution density on the genome, easy data integration, and high detection throughput. At the same time, the publication of a series of reference genome sequences, the release of a large amount of re-sequencing data, and the rapid development of high-throughput genotyping platforms have laid a good foundation for the application of SNP markers in the field of maize variety identification. Up to now, the SNP marker loci developed and screened for crop variety identification are all bi-allelic variant loci, that is, each locus has 2 allelic variants and 3 genotypes. For the bi-allelic variant type of SNP markers, the polymorphism of a single locus is relatively low; at the same time, due to only 2 variants and 3 genotypes, it has a certain impact on data reading for variety identification, especially in the case of detecting mixed samples; at the same time, there are also cases of multi-allelic variants for SNP loci associated with traits in addition to bi-allelic variants. In the maize genome, in addition to the bi-allelic variant SNP type with the highest distribution density, there are also tri-allelic and tetra-allelic SNP loci. For each additional allelic variant at a single locus, its polymorphism increases exponentially. For example, tri-allelic variant SNP markers can have 3 allelic variants and 6 genotypes. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a combination of maize tri-allelic variant SNP loci and its application.

[0005] The present invention collected 388 maize inbred line materials with wide sources, rich phenotypes and genotypes, and strong representativeness, and carried out whole-genome high-throughput sequencing. By using methods such as bioinformatics, statistical analysis, and targeted sequencing verification, a set of tri-allelic variant SNP core locus combinations containing 40 loci was finally determined, providing technical support for the authenticity, parentage, and purity identification of maize varieties.

[0006] In the first aspect, the present invention provides a tri-allelic SNP locus combination, including the following SNP loci: TaSNP01: chr1: 26694070: A / C / G, TaSNP02: chr1: 70264325: C / A / G, TaSNP03: chr1: 70370458: C / T / G, TaSNP04: chr1: 268027869: T / C / A, TaSNP05: chr2: 56077965: T / A / G, TaSNP06: chr2: 191937511: T / A / C, TaSNP07: chr2: 192091927: A / T / C, TaSNP08: chr2: 237697548: G / T / C, TaSNP09: chr3: 47899705: G / A / C, TaSNP10: chr3: 129111971: A / G / T, TaSNP11: chr3: 222001675: C / A / T, TaSNP12: chr3: 224178922: C / G / A, TaSNP13: chr4: 3788094: G / A / T, TaSNP14: chr4: 10076723: G / C / T, TaSNP15: chr4: 10645740: A / C / G, TaSNP16: chr4: 86481785: C / A / T, TaSNP17: chr5: 1487478: T / G / A, TaSNP18: chr5: 60276448: G / C / T, TaSNP19: chr5: 91897631: G / A / T, TaSNP20: chr5: 186888339: C / T / G, TaSNP21: chr6: 75706844: T / G / A, TaSNP22: chr6: 149861566: T / G / C, TaSNP23: chr6: 151920452: G / T / A, TaSNP24: chr6: 167615957: C / A / G, TaSNP25: chr7: 20793475: A / T / G, TaSNP26: chr7: 23775501: T / G / A, TaSNP27: chr7: 25104386: T / A / C, TaSNP28: chr7: 91528461: G / T / A, TaSNP29: chr8: 117427483: G / T / C, TaSNP30: chr8: 141530330: A / C / G, TaSNP31: chr8: 141766718: C / G / T, TaSNP32: chr8: 172879909: A / G / C, TaSNP33: chr9: 46962286: T / C / A, TaSNP34: chr9: 88827030: T / C / A, TaSNP35: chr9: 97080329: A / T / CTaSNP36: chr9:149325574: A / G / C, TaSNP37: chr10:16222443: A / T / G, TaSNP38: chr10:113185518: T / G / C, TaSNP39: chr10:145237355: C / G / A, TaSNP40: chr10:146434632: G / C / T.

[0007] The SNP locus format described in the present invention is a commonly used expression method in the art. Taking TaSNP01: chr1:26694070: A / C / G as an example, TaSNP01 is the name of the SNP locus, chr1 indicates that it is located on chromosome 1, 26694070 indicates that it is located at the 26694070th position on the chromosome, and A / C / G indicates the polymorphism of this SNP locus.

[0008] In a second aspect, the present invention provides a combination of tri-allelic SNP loci, including: the nucleotide sequences shown in SEQ ID NO.1-40; The nucleotide sequence shown in SEQ ID NO.1 has polymorphism at position 61, and the polymorphism is A / C / G, The nucleotide sequence shown in SEQ ID NO.2 has polymorphism at position 61, and the polymorphism is C / A / G, The nucleotide sequence shown in SEQ ID NO.3 has polymorphism at position 61, and the polymorphism is C / T / G, The nucleotide sequence shown in SEQ ID NO.4 has polymorphism at position 61, and the polymorphism is T / C / A, The nucleotide sequence shown in SEQ ID NO.5 has polymorphism at position 61, and the polymorphism is T / A / G, The nucleotide sequence shown in SEQ ID NO.6 has polymorphism at position 61, and the polymorphism is T / A / C, The nucleotide sequence shown in SEQ ID NO.7 has polymorphism at position 61, and the polymorphism is A / T / C, The nucleotide sequence shown in SEQ ID NO.8 has polymorphism at position 61, and the polymorphism is G / T / C, The nucleotide sequence shown in SEQ ID NO.9 has polymorphism at position 61, and the polymorphism is G / A / C, The nucleotide sequence shown in SEQ ID NO.10 has polymorphism at position 61, and the polymorphism is A / G / T, The nucleotide sequence shown in SEQ ID NO.11 has polymorphism at position 61, and the polymorphism is C / A / T, The nucleotide sequence shown in SEQ ID NO.12 has a polymorphism at position 61, and the polymorphism is C / G / A. The nucleotide sequence shown in SEQ ID NO.13 has a polymorphism at position 61, and the polymorphism is G / A / T. The nucleotide sequence shown in SEQ ID NO.14 has a polymorphism at position 61, and the polymorphism is G / C / T. The nucleotide sequence shown in SEQ ID NO.15 has a polymorphism at position 61, and the polymorphism is A / C / G. The nucleotide sequence shown in SEQ ID NO.16 has a polymorphism at position 61, and the polymorphism is C / A / T. The nucleotide sequence shown in SEQ ID NO.17 has a polymorphism at position 61, and the polymorphism is T / G / A. The nucleotide sequence shown in SEQ ID NO.18 has a polymorphism at position 61, and the polymorphism is G / C / T. The nucleotide sequence shown in SEQ ID NO.19 has a polymorphism at position 61, and the polymorphism is G / A / T. The nucleotide sequence shown in SEQ ID NO.20 has a polymorphism at position 61, and the polymorphism is C / T / G. The nucleotide sequence shown in SEQ ID NO.21 has a polymorphism at position 61, and the polymorphism is T / G / A. The nucleotide sequence shown in SEQ ID NO.22 has a polymorphism at position 61, and the polymorphism is T / G / C. The nucleotide sequence shown in SEQ ID NO.23 has a polymorphism at position 61, and the polymorphism is G / T / A. The nucleotide sequence shown in SEQ ID NO.24 has a polymorphism at position 61, and the polymorphism is C / A / G. The nucleotide sequence shown in SEQ ID NO.25 has a polymorphism at position 61, and the polymorphism is A / T / G. The nucleotide sequence shown in SEQ ID NO.26 has a polymorphism at position 61, and the polymorphism is T / G / A. The nucleotide sequence shown in SEQ ID NO.27 has a polymorphism at position 61, and the polymorphism is T / A / C. The nucleotide sequence shown in SEQ ID NO.28 has a polymorphism at position 61, and the polymorphism is G / T / A. The nucleotide sequence shown in SEQ ID NO.29 has a polymorphism at position 61, and the polymorphism is G / T / C. The nucleotide sequence shown in SEQ ID NO.30 has a polymorphism at position 61, and the polymorphism is A / C / G. The nucleotide sequence shown in SEQ ID NO.31 has a polymorphism at position 61, and the polymorphism is C / G / T. The nucleotide sequence shown in SEQ ID NO.32 has a polymorphism at position 61, and the polymorphism is A / G / C. The nucleotide sequence shown in SEQ ID NO.33 has a polymorphism at position 61, and the polymorphism is T / C / A. The nucleotide sequence shown in SEQ ID NO.34 has a polymorphism at position 61, and the polymorphism is T / C / A. The nucleotide sequence shown in SEQ ID NO.35 has a polymorphism at position 61, and the polymorphism is A / T / C. The nucleotide sequence shown in SEQ ID NO.36 has a polymorphism at position 61, and the polymorphism is A / G / C. The nucleotide sequence shown in SEQ ID NO.37 has a polymorphism at position 61, and the polymorphism is A / T / G. The nucleotide sequence shown in SEQ ID NO.38 has a polymorphism at position 61, and the polymorphism is T / G / C. The nucleotide sequence shown in SEQ ID NO.39 has a polymorphism at position 61, and the polymorphism is C / G / A. The nucleotide sequence shown in SEQ ID NO.40 has a polymorphism at position 61, and the polymorphism is G / C / T.

[0009] In a third aspect, the present invention provides a molecular probe, which is used to detect the aforementioned SNP locus combination.

[0010] In a fourth aspect, the present invention provides a gene chip, which includes the aforementioned molecular probe.

[0011] In a fifth aspect, the present invention provides a kit, which includes the aforementioned SNP locus combination, or the aforementioned molecular probe, or the aforementioned gene chip.

[0012] In a sixth aspect, the present invention provides the application of the aforementioned SNP locus combination as a target in the preparation of a gene chip for maize variety identification, genetic relationship identification, purity identification or excellent germplasm selection.

[0013] The present invention further provides the application of the aforementioned SNP locus combination, or the aforementioned molecular probe, or the aforementioned gene fragment, or the aforementioned kit in maize variety identification, genetic relationship identification or purity identification.

[0014] Further, the variety identification includes: Detecting the polymorphism of the SNP loci as described above for the maize sample to be tested, comparing the polymorphism detection results with the standard genotype map, and judging the variety type of the maize sample to be tested according to the comparison results.

[0015] The standard genotype map of the present invention is a map composed of polymorphism data of known maize varieties at these 40 known SNP loci. Specifically, in the actual production process, the polymorphism of the maize sample to be tested at these 40 SNP loci can be compared with the map to identify the maize variety with the highest coincidence degree. In addition, the polymorphism of the maize sample to be tested and the known maize sample at these 40 SNP loci can also be detected simultaneously, and it can be judged whether the maize sample to be tested and the known maize sample belong to the same variety according to whether the repeatability (the proportion of the same loci) meets the requirements (the threshold can be set by oneself).

[0016] Further, the genetic relationship identification includes: Detecting the polymorphism of the SNP loci as described above for the suspected genetically related maize sample combination, analyzing the polymorphism results of all maize samples in the suspected genetically related maize sample combination, and judging the genetic relationship of each maize sample in the suspected genetically related maize sample combination according to the analysis results.

[0017] The present invention further provides the application of the foregoing SNP locus combination as a target in the breeding of excellent maize germplasm.

[0018] The present invention has the following beneficial effects: The present invention has screened and obtained a triple-allele variant SNP locus combination for maize breeding. Using this SNP locus combination, variety identification, genetic relationship identification and purity identification can be carried out for maize materials, and it has high resolution and accuracy at the same time.

[0019] The triple-allele variant SNP locus combination provided by the present invention is beneficial to improving the authenticity of varieties and the efficiency of paternity identification, reducing costs; is beneficial to identifying abnormal plants in seed purity, and is beneficial to solving the problem of accurate reading of genotype data caused by using mixed plants to prepare test samples. The triple-allele variant SNP provided by the present invention expands the range of available marker loci of maize at the genomic level, and provides new ideas and methods for research such as maize variety and germplasm resource identification and genetic relationship evaluation. Description of the Drawings

[0020] To more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0021] Figure 1 It is a comparison of the effects of 40 three - allele variant SNP locus combinations and 96 two - allele variant SNP locus combinations provided by the present invention. Detailed implementation manners

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are some embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0023] For the experimental methods involved in the following embodiments, unless otherwise specified, they are all conventional methods in the art. For example, reference can be made to the experimental manuals in the art or the conditions recommended in the manufacturer's instructions.

[0024] For the experimental materials and reagents involved in the following embodiments, unless otherwise specified, they can all be obtained through commercial channels.

[0025] Example 1 The present invention provides 40 three - allele variant SNP loci, which are mined through the following methods: (1) Whole - genome re - sequencing data: 388 maize inbred lines with wide global representation were selected for whole - genome re - sequencing to obtain high - quality second - generation sequencing data fragments.

[0026] (2) Total initial variant loci: The BWA and GATK software were used for sequence alignment and variant mining, and a total of 780K initial multi - allele variant SNP loci (789553) were obtained, including three - allele, four - allele, and five - allele types of loci.

[0027] (3) Elite locus pool: Based on the sequencing data coverage (≥50%) and the Quality value (Q value ≥ 50000) of each locus evaluated by the GATK software, a preliminary screening of loci was carried out, and a total of 70K multi - allele variant SNP loci (72830) were obtained to form an elite locus set.

[0028] (4) Candidate locus set: Based on the sequencing data, the genotype data of 388 inbred lines were extracted to evaluate the heterozygosity rate and polymorphism of loci. Loci with a heterozygosity rate > 0.4 were deleted, and loci with a PIC value < 0.1 were deleted; multi-copy loci were deleted; loci with ≥2 InDels (insertion-deletion > 2bp) in the flanking sequences were deleted; candidate loci were uniformly selected across the whole genome based on the physical and genetic maps, and a total of 28,000 loci were obtained to form a candidate locus set. Among them, the genetic positions were evaluated and deleted according to more than 6,000 BINs divided by the maize genome.

[0029] (5) Design probes to obtain a set of multi-allelic SNP marker sets: Since the marker loci are multi-allelic variations, a sequencing platform should be used as the genotyping platform. Loci with a GC content value of the flanking 100bp sequence close to 50 were selected according to the requirements of the targeted sequencing genotyping platform, and the probe sequences of each locus were optimized to form a set of multi-allelic SNP marker sets, with the number of loci being 1,536.

[0030] (6) Design and synthesize probes for the above 1,536 loci based on the targeted sequencing technology, and use 200 national-approved hybrid varieties and 800 representative inbred lines for genotyping analysis on the sequencing platform. Strict evaluations were carried out respectively according to parameters such as the sequencing data coverage, data missing rate, heterozygous genotype frequency, PIC value, and uniform distribution of inbred lines and hybrid varieties, and a set of 40 loci with tri-allelic variations SNPs was determined (as shown in the following table, genome version number: B73 AGP_v3 reference).

[0031] Basic characteristics of 40 loci: Based on the evaluation of 200 hybrid varieties and 800 inbred lines, the results showed that: (1) There was no data missing in the hybrid varieties, and the average missing rate in the inbred lines was 0.10%. (2) The average heterozygous genotype frequency in the hybrid varieties was 61.19%, with a distribution range of 48.39% - 72.35%; the average heterozygous genotype frequency in the inbred lines was 6.62%, with a range of 4.98% - 7.93%. (3) The average PIC value (polymorphism information content) of the loci in the hybrid varieties was 0.47, with a range of 0.40 - 0.55; the average PIC value of the loci in the inbred lines was 0.48, with a range of 0.46 - 0.51.

[0032] As shown in the above table are the 40 tri-allelic SNP loci disclosed by the present invention, including chromosome loci, their physical positions, allelic variations, and flanking sequences (the nucleotide sequences on both sides of the SPN locus, including the locus itself).

[0033] Compared with the diallel SNP loci, the 40 tri-allelic SNP loci provided by the present invention have significantly improved ability to distinguish different varieties. The difference distribution of 200 nationally approved corn hybrids was analyzed by using 40 tri-allelic SNP loci and 96 di-allelic SNP loci (Tian Hongli et al., 2024, Acta Agronomica Sinica, 50:1115-1123). The results are as follows Figure 1 As shown, Figure 1 The horizontal axis is the percentage of differential sites, and the vertical axis is the logarithm of the pairwise comparison. The results of the 40 tri-allelic SNP loci combined to evaluate the hybrids showed that the percentage distribution of differential sites was mainly concentrated in the 50%-90% range, with the highest value area being around 70%; the results of the 96 di-allelic SNP loci combined to evaluate the hybrids showed that the percentage distribution of differential sites was mainly concentrated in the 40%-80% range, with the highest value area being around 60%.

[0034] The above results show that the 40 tri-allelic SNP loci showed higher resolution.

[0035] Example 2 Authenticity identification of maize varieties using the tri-allelic SNP core site combination provided in Example 1 This example identifies whether the corn sample to be tested (codenamed A) is a known reference corn variety Jingke 968 (codenamed B), that is, the authenticity of corn sample A. The specific identification process is as follows: 1. DNA extraction and quality identification The two samples of the tested corn sample (codenamed A) and the control corn variety Jingke 968 (codenamed B) were germinated with seeds, exposed to light, and formed green seedlings. The total genomic DNA of samples A and B was extracted using the conventional CTAB method. The DNA was diluted to form a working solution with a concentration of 20 ng / μL.

[0036] 2. Experimental process Primer design and synthesis of 40 polymorphic sites: The 40 polymorphic sites provided in this embodiment are all tri-allelic SNP markers, and the genotype can be detected using first-generation or second-generation high-throughput sequencing technology; the three allelic variations can also be designed as amplified fragments of different lengths, and the genotype can be detected using a fluorescent capillary electrophoresis platform. The principle of designing primers based on the electrophoresis platform is: three upstream primers are designed, and the 3' end of each primer is a specific allelic variation, and the three primers are designed with different lengths to distinguish different allelic variations; the downstream primer is a universal primer and is labeled with a fluorescent group at the 5' end.

[0037] PCR amplification system: The PCR reaction system was 20 μL, including 3 μL DNA, 0.25 μL 20 μmol / L primer, 10 μL 2×PCRmix, and 6.75 μL deionized water.

[0038] PCR amplification procedure: 95°C for 5 min; 95°C for 40 sec, 60°C for 35 s, 72°C for 45 s, 35 cycles; 72°C for 10 min, store at 4°C.

[0039] Electrophoresis: The PCR products were electrophoresed on an AB 3730XL DNA analyzer (Applied Biosystems, USA), a fluorescence capillary electrophoresis system. The PCR products, formamide, and internal standard (GeneScanTM-500 LIZ, Applied Biosystems, USA) were added to individual wells of a 96-well electrophoresis plate. The above mixed samples were run on a PCR instrument at 95°C for 5 min for denaturation. After taking out the denatured electrophoresis products, they were centrifuged at 1000 rpm / min for 1 min and then electrophoresed on the AB 3730XL DNA analyzer.

[0040] Genotype data acquisition: The original data was collected using the Date Collection Ver.1.0 software supporting the electrophoresis instrument. The original data was imported into the SSR Analyzer software for analysis to obtain the genotype data of each locus of samples A and B (Table 3).

[0041] Data comparison and analysis: The genotype data of 40 loci of the sample to be tested A and sample B were compared locus by locus. The results showed that there were 12 identical loci and 28 different loci between the two, and the proportion of identical loci was 30%. It was determined that the sample to be tested A was not Jingke 968.

[0042] The authenticity identification was carried out using the industry standard (NY / T 1432-2014, Technical Regulations for Maize Variety Identification - SSR Marker Method) of 40 SSR core loci currently in use, further confirming that the sample to be tested A was not Jingke 968.

[0043] Table 3 Genotype data of the maize sample to be tested A and the control maize sample B (Jingke 968)

[0044] The above results illustrate that the SNP locus combination provided by the present application can be used to identify the authenticity of maize varieties, based on the proportion of identical loci (self-set threshold).

[0045] Example 3 Identification of the parent-child relationship of maize varieties using a set of three-allele variant SNP core locus combinations provided in Example 1 The present invention identified whether there was a parent-child relationship between the maize sample to be tested (code named C) and the maize hybrids Xianyu 335 (code named D) and Zhengdan 958 (code named E). The specific identification process was as follows: 1. Extract DNA from the maize samples Three samples, namely the maize sample to be tested (coded as C), the maize hybrid Xianyu 335 (coded as D), and Zhengdan 958 (coded as E), were germinated from seeds, given light, and green seedlings were formed. The total genomic DNA of samples A and B was extracted using the conventional CTAB method. The DNA was diluted to form a working solution with a concentration of 20 ng / μL.

[0046] 2. Experimental procedure The primer design and synthesis, PCR amplification system, PCR amplification program, and electrophoresis for 40 polymorphic loci were the same as in Example 2.

[0047] Obtaining genotype data: The original data was collected using the Date Collection Ver.1.0 software supporting the electrophoresis instrument, and the original data was imported into the SSR Analyzer software for analysis to obtain the genotype data of each locus for samples C, D, and E (Table 4).

[0048] Data comparison and analysis: The genotype data of 40 loci of the sample C to be tested was compared with that of samples D (Xianyu 335) and E (Zhengdan 958) locus by locus. The results showed that there were 15 loci excluding the parent - child relationship between sample C and sample D (Xianyu 335), and 0 loci excluding the parent - child relationship between sample C and sample E (Zhengdan 958). It was determined that the sample C to be tested excluded the parent - child relationship with sample D (Xianyu 335), and did not exclude the parent - child relationship with sample E (Zhengdan 958). Comparing sample C with the known inbred line database, it was confirmed to be the inbred line sample Zheng 58, that is, the female parent of the maize hybrid Zhengdan 958.

[0049] Table 4 Genotype data of the maize inbred line sample C to be tested, hybrid Xianyu 335, and hybrid Zhengdan 958

[0050] Thus, it can be seen that the 40 three - allele SNP loci provided by the present invention can accurately identify the parent - child relationship of maize varieties. This shows that the 40 three - allele SNP loci provided by the present invention can be further applied in the following aspects: (1) Accurately identify the genetic relationship of varieties to verify the authenticity of hybrid offspring and avoid breeding failures caused by parental mixing or self - crossing.

[0051] (2) Ensure the genetic consistency of commercial seeds and prevent the impact of seed mixing on yield and resistance.

[0052] (3) Conduct genetic diversity analysis on maize germplasm resources and optimize resource preservation and utilization strategies.

[0053] The three - allele SNP loci provided by the present invention have the advantages of high accuracy and stability, and have high market and industrial value.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tri-allelic variation SNP site combination, characterized in that: The following SNP sites are included: TaSNP01:chr1:26694070:A / C / G、TaSNP02:chr1:70264325:C / A / G、TaSNP03:chr1:70370458:C / T / G、TaSNP04:chr1:268027869:T / C / A、TaSNP05:chr2:56077965:T / A / G、TaSNP06:chr2:191937511:T / A / C、TaSNP07:chr2:192091927:A / T / C、TaSNP08:chr2:237697548:G / T / C、TaSNP09:chr3:47899705:G / A / C、TaSNP10:chr3:129111971:A / G / T、TaSNP11:chr3:222001675:C / A / T、TaSNP12:chr3:224178922:C / G / A、TaSNP13:chr4:3788094:G / A / T、TaSNP14:chr4:10076723:G / C / T、TaSNP15:chr4:10645740:A / C / G、TaSNP16:chr4:86481785:C / A / T、TaSNP17:chr5:1487478:T / G / A、TaSNP18:chr5:60276448:G / C / T、TaSNP19:chr5:91897631:G / A / T、TaSNP20:chr5:186888339:C / T / G、TaSNP21:chr6:75706844:T / G / A、TaSNP22:chr6:149861566:T / G / C、TaSNP23:chr6:151920452:G / T / A、TaSNP24:chr6:167615957:C / A / G、TaSNP25:chr7:20793475:A / T / G、TaSNP26:chr7:23775501:T / G / A、TaSNP27:chr7:25104386:T / A / C、TaSNP28:chr7:91528461:G / T / A、TaSNP29:chr8:117427483:G / T / C、TaSNP30:chr8:141530330:A / C / G、TaSNP31:chr8:141766718:C / G / T、TaSNP32:chr8:172879909:A / G / C、TaSNP33:chr9:46962286:T / C / A、TaSNP34:chr9:88827030:T / C / A、TaSNP35:chr9:97080329:A / T / C、TaSNP36:chr9:149325574:A / G / C、TaSNP37:chr10:16222443:A / T / G、TaSNP38:chr10:113185518:T / G / C、TaSNP39:chr10:145237355:C / G / A、TaSNP40:chr10:146434632:G / C / T。、 2. A tri-allelic variant SNP site combination, characterized in that: include: The nucleotide sequence shown in SEQ ID NO.1-40; As shown in SEQ ID NO.1, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is A / C / G. As shown in SEQ ID NO.2, the 61st position of the nucleotide sequence has a polymorphism, and the polymorphism is C / A / G. As shown in SEQ ID NO.3, the 61st position of the nucleotide sequence has a polymorphism, and the polymorphism is C / T / G. As shown in SEQ ID NO.4, the 61st position of the nucleotide sequence has a polymorphism, and the polymorphism is T / C / A. As shown in SEQ ID NO.5, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is T / A / G. As shown in SEQ ID NO.6, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is T / A / C. As shown in SEQ ID NO.7, the 61st position of the nucleotide sequence has a polymorphism, and the polymorphism is A / T / C. As shown in SEQ ID NO.8, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is G / T / C. As shown in SEQ ID NO.9, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is G / A / C. As shown in SEQ ID NO.10, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is A / G / T. As shown in SEQ ID NO.11, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is C / A / T. As shown in SEQ ID NO.12, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is C / G / A. As shown in SEQ ID NO.13, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is G / A / T. As shown in SEQ ID NO.14, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is G / C / T. As shown in SEQ ID NO.15, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is A / C / G. As shown in SEQ ID NO.16, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is C / A / T. As shown in SEQ ID NO.17, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is T / G / A. As shown in SEQ ID NO.18, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is G / C / T. As shown in SEQ ID NO.19, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is G / A / T. As shown in SEQ ID NO.20, the 61st position of the nucleotide sequence has a polymorphism, and the polymorphism is C / T / G. As shown in SEQ ID NO.21, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is T / G / A. As shown in SEQ ID NO.22, the 61st position of the nucleotide sequence has a polymorphism, and the polymorphism is T / G / C. As shown in SEQ ID NO.23, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is G / T / A. As shown in SEQ ID NO.24, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is C / A / G. As shown in SEQ ID NO.25, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is A / T / G. As shown in SEQ ID NO.26, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is T / G / A. As shown in SEQ ID NO.27, the 61st position of the nucleotide sequence has a polymorphism, and the polymorphism is T / A / C. As shown in SEQ ID NO.28, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is G / T / A. As shown in SEQ ID NO.29, the 61st position of the nucleotide sequence has a polymorphism, and the polymorphism is G / T / C. As shown in SEQ ID NO.30, the 61st position of the nucleotide sequence has a polymorphism, and the polymorphism is A / C / G. As shown in SEQ ID NO.31, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is C / G / T. As shown in SEQ ID NO.32, the 61st position of the nucleotide sequence has a polymorphism, and the polymorphism is A / G / C. As shown in SEQ ID NO.33, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is T / C / A. As shown in SEQ ID NO.34, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is T / C / A. As shown in SEQ ID NO.35, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is A / T / C. As shown in SEQ ID NO.36, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is A / G / C. As shown in SEQ ID NO.37, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is A / T / G. As shown in SEQ ID NO.38, the 61st position of the nucleotide sequence has a polymorphism, and the polymorphism is T / G / C. As shown in SEQ ID NO.39, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is C / G / A. As shown in SEQ ID NO.40, the nucleotide sequence at position 61 has a polymorphism, and the polymorphism is G / C / T.

3. A molecular probe, characterized in that The molecular probe is used to detect the SNP site combination described in claim 1 or 2.

4. A gene chip, characterized in that: The gene chip comprises the molecular probe according to claim 3.

5. A kit, characterized in that: The kit comprises the SNP site combination according to claim 1 or 2, or the molecular probe according to claim 3, or the gene chip according to claim 4.

6. Use of the SNP site combination according to claim 1 or 2 as a target in the preparation of a gene chip for corn variety identification, kinship identification, purity identification or excellent germplasm breeding.

7. Use of the SNP site combination according to claim 1 or 2, or the molecular probe according to claim 3, or the gene fragment according to claim 4, or the kit according to claim 5 in corn variety identification, kinship identification or purity identification.

8. The use according to claim 7, characterized in that: The variety identification includes: The polymorphism of the SNP site as claimed in claim 1 or 2 is detected for the corn sample to be tested, the polymorphism detection result is compared with the standard genotype map, and the variety type of the corn sample to be tested is determined according to the comparison result.

9. The use according to claim 7, characterized in that: The kinship identification includes: The polymorphism of the SNP loci as described in claim 1 or 2 is detected for the suspected related corn sample combination, the polymorphism results of all corn samples in the suspected related corn sample combination are analyzed, and the relationship of each corn sample in the suspected related corn sample combination is determined according to the analysis results.

10. Use of the SNP site combination according to claim 1 or 2, or the molecular probe according to claim 3, or the gene fragment according to claim 4, or the kit according to claim 5 in the breeding of excellent corn germplasm.

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