Molecular marker of resistance-related gene of corn leaf beetle on chromosome 3 and application thereof

Abstract

The application discloses a molecular marker of a corn chromosome 3 leaf mite resistance related gene and application thereof. The application provides application of a substance for detecting the genotype of a SNP site Zm000011d042333@161,630,118 in a corn genome in at least one of the following: A1) identifying or assisting in identifying corn leaf mite resistance; A2) breeding corn with high leaf mite resistance; the SNP site Zm000011d042333@161,630,118 is the 26th in sequence 1. The application first discovers the gene Zm000011d042333 related to mite resistance in corn, and identifies the mite resistance of corn according to the genotype of the SNP site on the gene, and designs a KASP marker, which can quickly and accurately determine the genotype of the SNP site, and effectively screen corn resistant plants according to the genotype, and accelerate corn resistance breeding.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to a molecular marker of a corn chromosome 3 spider mite resistance related gene and application thereof. BACKGROUND

[0002] Tetranychus urticae Koch, is one of the most destructive agricultural pests in the world, which can harm more than 1000 plants including corn. Hot and dry climatic conditions are usually conducive to the rapid proliferation of spider mite populations. Due to the relatively weak resistance of corn germplasm to mites, combined with the continuous drought climate in the northwest region, spider mites have become one of the most important pests in the northwest corn production area.

[0003] So far, pesticides have been the main measure to control the damage of spider mites. Although the use of insecticides has a significant effect, long-term overuse has led to a series of environmental and ecological problems, such as the generation of pesticide resistance, pesticide residues and pest resurgence. Predatory mites can be an ideal biological control method for spider mites, but the high cost and long control cycle of natural enemies still cannot meet the actual production needs in the field. In addition, Bt transgenic corn has resistance to lepidopteran pests, but has no control effect on spider mites, and the large-area planting of Bt corn varieties may change the ecological niche of spider mites and increase the uncertainty of its prevalence. Therefore, it is of great significance to determine the resistance source of corn spider mite germplasm and understand the genetic mechanism of corn mite resistance for corn breeding and sustainable agricultural development.

[0004] The source of spider mite resistance has been reported in American materials, for example, in vitro leaf experiments have confirmed that inbred line Oh43 has strong resistance to spider mites. In addition, inbred line B96 and its derived lines B49 and B75 show strong resistance to spider mites, and gene mapping shows that a large-effect QTL on chromosome 6 controls spider mite resistance. In addition, previous studies have shown that China's Huangzao four improved inbred line Jing92 has high resistance to spider mites at the seedling and mature stages.

[0005] However, the molecular mechanism of corn mite resistance is still unclear, especially in Chinese inbred lines, no gene locus related to mite resistance has been identified so far, and no molecular marker associated with it has been discovered. Therefore, identifying QTL, candidate genes in Jing92 and developing molecular markers have important roles in the application of marker-assisted selection in breeding practice, which will greatly accelerate the creation and selection of new corn varieties with high mite resistance. SUMMARY

[0006] The purpose of the present application is to provide a molecular marker of a corn chromosome 3 spider mite resistance related gene and application thereof.

[0007] In a first aspect, the present invention provides a substance for detecting the genotype of the SNP locus Zm000011d042333@161,630,118 in the maize genome in at least one of the following applications:

[0008] A1) Identification or auxiliary identification of corn spider mite resistance;

[0009] A2) Breed maize with high resistance to spider mites;

[0010] The SNP site Zm000011d042333@161,630,118 is the 26th position of sequence 1.

[0011] In the application described above, the genotype of the SNP site Zm000011d042333@161,630,118 is GG, AA, or AG.

[0012] In the application described above, the substance used to detect the SNP site Zm000011d042333@161,630,118 in the maize genome is 1) or 2):

[0013] 1) Primer set;

[0014] 2) PCR reagents or kits containing the aforementioned primer set;

[0015] The primer set includes primer 1, primer 2 and primer 3;

[0016] The nucleotide sequence of primer 1 includes the sequence shown in sequence 2;

[0017] The nucleotide sequence of primer 2 includes the sequence shown in sequence 3;

[0018] The nucleotide sequence of primer 3 is sequence 4.

[0019] Primer 1 above is an upstream primer with a fluorescent sequence added to the 5' end of the upstream primer shown in sequence 2; in the embodiment of the present invention, a specific fluorescent sequence FAM is added: 5'-GAAGGTGACCAAGTTCATGCT-3', to obtain an upstream primer with the specific fluorescent sequence FAM added to the 5' end;

[0020] Primer 2 above is obtained by adding another fluorescent sequence to the 5' end of the upstream primer shown in sequence 3; in the embodiment of the present invention, the specific fluorescent sequence HEX is added: F5'-GAAGGTCGGAGTCAACGGATT-3', resulting in an upstream primer with the specific fluorescent sequence HEX added to the 5' end.

[0021] In a second aspect, the present invention provides any of the following substances:

[0022] The primer set described in the first aspect;

[0023] or, a PCR reagent or kit containing the set of primers of the first aspect.

[0024] In a third aspect, the present application provides use of the set of primers, the PCR reagent or the kit of the second aspect in at least one of the following:

[0025] A1) identifying or assisting in identifying corn spider mite resistance;

[0026] A2) breeding corn with high spider mite resistance.

[0027] In a fourth aspect, the present application provides a method for identifying or assisting in identifying corn spider mite resistance, comprising the following steps: detecting the genotype of the SNP site Zm000011d042333@161,630,118 in the corn genome as described in the first aspect, and making the following judgment:

[0028] Corn with the genotype of SNP Zm00001d042333@161,630,118 site as GG has greater or candidate greater spider mite resistance than corn with the genotype of SNP Zm00001d042333@161,630,118 site as AA or AG.

[0029] In the above method, the detection of the genotype of the SNP site Zm000011d042333@161,630,118 in the corn genome as described in the first aspect is a KASP reaction of the set of primers of the second aspect on the corn genome, and the obtained PCR amplification product is genotyped.

[0030] The genotyping method described above is that after irradiation by a fluorescence enzyme label instrument, if the PCR product only shows the color of the 5' end of the DNA molecule connected to the fluorescent sequence as shown in sequence 2, then the genotype of Zm00001d042333@161,630,118 site in the corn genome to be tested is G:G; if the PCR product only shows the color of the 5' end of the DNA molecule connected to the fluorescent sequence as shown in sequence 3, then the genotype of Zm00001d042333@161,630,118 site in the corn genome to be tested is A:A;

[0031] If the PCR product shows the color of the 5' end of the DNA molecule connected to the fluorescent sequence as shown in sequence 2 and the color of the 5' end of the DNA molecule connected to the fluorescent sequence as shown in sequence 3, then the genotype of Zm00001d042333@161,630,118 site in the corn genome to be tested is A:G.

[0032] In a fifth aspect, the present application provides a method for breeding corn with high resistance to spider mites, comprising the following steps: selecting corn with genotype GG at the SNP Zm00001d042333@161,630,118 site in the method of the fourth aspect for breeding, and obtaining the target corn.

[0033] The corn is a RIL population of Jing 92 and Jing 2416 or offspring thereof, and in the embodiments of the present application, F2 generations of the spider mite-resistant single plant and the spider mite-sensitive single plant in the RIL population of Jing 92 and Jing 2416 are used; specifically, F2 population of RIL 007 (resistant, spider mite level is two) * RIL 009 (sensitive, spider mite level is eight) or F2 population of RIL 284 (resistant, spider mite level is two) * RIL 004 (sensitive, spider mite level is eight) in the RIL population of Jing 92 and Jing 2416.

[0034] The present application first discovers the gene Zm000011d042333 related to resistance to spider mites in corn, and identifies the resistance to spider mites of corn according to the genotype of the SNP site on the gene, which creates a basis for breeding and screening resistance corn breeding resources. According to the site, a KASP marker is designed, which can quickly and accurately determine the genotype of the SNP site, and effectively screen corn resistant plants according to the genotype, and accelerate the breeding of corn resistance. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Phenotype identification of Jing 92 (J92) * Jing 2416 (J2416) population resistance to spider mites. A is the picture of the resistance of corn leaves to spider mites; B is the resistance rating level of leaves in Hainan in 21 (21HN) and Beijing Tongzhou in 22 (22TZ); C is the distribution of the rating level of spider mite damage of corn single plant leaves in Hainan in 21 and Beijing Tongzhou in 22; D is the correlation of resistance rating in Hainan in 21 and Beijing Tongzhou in 22.

[0036] Figure 2 QTL positioning of spider mite phenotype. A is the positioning of the resistance to spider mites trait on chromosome 3 in Hainan in 2021 and Beijing Tongzhou in 2022; B is all the QTL sites preliminarily positioned. C is the interval for fine positioning according to 20 SNPs last year.

[0037] Figure 3 KASP genotyping result chart of the candidate gene in the RIL 007 x RIL 009 population and the corresponding spider mite resistance phenotype column chart. A is the KASP genotyping result chart of the Zm00001d042333 gene; B is the column chart of the field spider mite resistance level of different Zm00001d042333 genotypes.

[0038] Figure 4Figure of KASP genotyping results of candidate genes in RIL004 x RIL284 population and corresponding leaf mite resistance phenotype column chart. A is the figure of KASP genotyping results of Zm00001d042333 gene; B is the column chart of field leaf mite resistance grade of different Zm00001d042333 genotypes. DETAILED DESCRIPTION

[0039] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0040] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0041] The application will be further described in detail below with reference to the specific embodiments, and the examples given are only to illustrate the application, not to limit the scope of the application.

[0042] In the following examples, the KASP reagent contains three parts, sample DNA, KASP TM Components primer and PCR system. Sample DNA is obtained by SDS method, KASP TM Components primer contains two forward primers and one reverse primer as KASP Assay mix. KASP Master mix contains a pair of FRET cassette, ROX reference dye, Klear Taq DNA polymerase, dNTP, buffer and Mgcl2, etc. For the FRET structure contained in the PCR reaction system, the fluorescent group has no fluorescence signal before reaction, and the fluorescent group produces fluorescence by two-step binding to the corresponding sequence during reaction. The size of corn genome: 2192.4 Mb, 10 ng / μl as the starting concentration of DNA for KASP experiment. The concentration of this test is 25 μmol / L (final concentration of system).

[0043] KASP amplification program: 61-55℃ two-step falling PCR program. Stage 1, 95℃ pre-denaturation for 15 min; stage 2, 94℃ denaturation for 20 s, 61-55℃ annealing for 1 min (decrease 0.6℃ per cycle, 10 cycles), stage 3, 94℃ denaturation for 20 s, 55℃ annealing for 1 min (32 cycles).

[0044] Figure 2: Genotyping data result reading: each data point represents a DNA sample. Samples with the same genotyping result are aggregated in one position. The x-axis, y-axis and diagonal line of the coordinate axis distinguish the genotyping results. The red mark close to the y-axis represents that the DNA sample is homozygous for the HEX marker allele, the blue mark close to the x-axis represents that the DNA sample is homozygous for the FAM marker allele, and the green mark on the diagonal line represents that the DNA sample is heterozygous, one for the FAM marker allele and one for the HEX marker allele.

[0045] Jing 2416 is described in the following document: Zhao Jiuren et al, Breeding and Application of Maize Inbred Line Jing 2416, Journal of Plant Genetic Resources, 2020.3.12;

[0046] Jing 92 is described in the following document: Chen Yafeng et al, Effects of Leaf Physical Traits on the Resistance of Maize Hybrid Jingke 968 to Tetranychus urticae Koch, Environmental Entomology, 2022, 44(1): 229-235.

[0047] Example 1: Obtaining of SNP markers of corn mite resistance related genes

[0048] I. Discovery of SNP sites of corn mite resistance related genes

[0049] Jing 2416 was used as the female parent and Jing 92 was used as the male parent to obtain F1, and then F1 was self-crossed to F2. One seed of each ear of F2 was selected and self-crossed, and this process was repeated until F6, which was the RIL population (this population was named J92*J2416 population).

[0050] The J92*J2416 population of 318 individuals was grown to the tasseling stage at two locations, Beijing Tongzhou in May 2022 and Hainan in October 2021, and the degree of corn mite damage was investigated after the mites were introduced to identify the mite resistance phenotype (see Di et al, 2014, Laboratory and field evaluation of maize resistance to the two-spotted spider mite, Tetranychus urticae).

[0051] The investigation standard for leaf mite feeding is as follows, and the grade division standard is shown in Table 1: the ear leaf is defined as level three damage by mite feeding, and is defined as level four damage when severe; the damage to the leaf above the ear leaf is defined as one level higher (for example, the leaf above the ear leaf is often defined as level four damage, and the two leaves above the ear leaf are often defined as level five damage), and generally 1-3 is strong mite resistance and 8-10 is mite sensitive. The higher the grade, the worse the mite resistance.

[0052] Table 1 is the leaf mite grade classification standard

[0053]

[0054] The two phenotypic data are significantly correlated, the mite resistance phenotype shows additive effect, and both phenotypes are normally distributed Figure 1

[0055] QTL mapping was performed on the mite resistance phenotype of the above two populations of J92*J2416 Figure 2 ), and a 15M region on chromosome 3 was found to be commonly located in both years, with a phenotypic contribution rate of 12.1% in Hainan in 2021 and 4.3% in Tongzhou in 2022, containing 237 genes. It is a new QTL site derived from J92, which is different from the resistance source of B96 (chromosome 6) of the American material.

[0056] Using RIL007 (resistant, leaf mite grade two) and RIL009 (sensitive, leaf mite grade eight) samples selected from the J92*J2416 population after mite resistance phenotype identification, an F2 population of 1468 strains was constructed after hybridization, 20 new markers were developed in the 15M region to fine map the mite resistance site, screen candidate genes, and anchor a nonsynonymous SNP site on the candidate gene Zm000011d042333. The site was determined by comparing the gene sequences of J92 and J2416. The nonsynonymous SNP is located at position 161,630,118 of the B73 reference genome (Zm-B73-REFERENCE-GRAMENE-4.0 February 2017), which is mutated from nonsynonymous G to A in mite resistance.

[0057] Therefore, the nonsynonymous SNP is named Zm000011d042333@161,630,118.

[0058] Zm000011d042333@161,630,118 is located at position 161,630,118 of the maize B73 reference genome or at position 26 of sequence 1, and the genotype of the SNP site is GG, AA or AG; sequence 1: TGCTTTCAGATTCCTGCGAGCAATGRTGAATGCATCTATTCT, R is G or A.

[0059] II. Establishment of method for detecting SNP Zm000011d042333@161,630,118

[0060] 1. Design and synthesis of KASP primers

[0061] ​To further verify the relationship between SNP site and the leaf-eating grade phenotype of spider mites, KASP analysis was performed on the possible functional SNP in the entire mapping population, and the primer was designed as follows:

[0062] According to the nucleotide sequences before and after sequence 1 of SNP Zm00001d042333@161,630,118, the KASP primer set for detecting SNP Zm00001d042333@161,630,118 site was designed (denoted as KASP molecular marker Zm00001d042333 (ZM-333)) as follows:

[0063] Upstream primer: F 5'-CTGAAACTATAAGAATAGATGCATTCAC-3' (sequence 2),

[0064] Upstream primer: F 5'-GTCTGAAACTATAAGAATAGATGCATTCAT-3' (sequence 3),

[0065] Downstream primer: R 5'-CTAATGCTTTCAGATTCCTGCGAGCAA-3' (sequence 4).

[0066] Add specific fluorescent sequence FAM to the 5' end of the upstream primer shown in sequence 2: 5'-GAAGGTGACCAAGTTCATGCT-3', to obtain the upstream primer with specific fluorescent sequence FAM added to the 5' end;

[0067] Add specific fluorescent sequence HEX to the 5' end of the upstream primer shown in sequence 3: F 5'-GAAGGTCGGAGTCAACGGATT-3', to obtain the upstream primer with specific fluorescent sequence HEX added to the 5' end.

[0068] The above-mentioned upstream primer with specific fluorescent sequence FAM added to the 5' end (sequence 2 after adding FAM) and the single-stranded DNA molecule shown in sequence 4 amplify the fragment of SNP Zm00001d042333@161,630,118 site genotype A:A, and the sequence carrying FAM after PCR amplification shows blue color under fluorescent irradiation;

[0069] The above-mentioned upstream primer with specific fluorescent sequence HEX added to the 5' end (sequence 3 after adding HEX) and the single-stranded DNA molecule shown in sequence 4 amplify the fragment of SNP Zm00001d042333@161,630,118 site genotype G:G, and the sequence carrying HEX after PCR amplification shows red color under fluorescent irradiation.

[0070] The upstream primer with the specific fluorescent sequence FAM added at the 5' end, the upstream primer with the specific fluorescent sequence HEX added at the 5' end and the single-stranded DNA molecule shown in sequence 4 are used to amplify the fragment of the SNP Zm00001d042333@161,630,118 site with the genotype A:G, and the product after PCR amplification shows green color under fluorescence irradiation.

[0071] The primer design of the KASP molecular marker Zm00001d042333 (ZM-333) is shown in Table 2, and the 142nd position of the sequence in the table is the SNP site:

[0072] Table 2 is the KASP molecular marker information

[0073]

[0074]

[0075] 2. KASP reaction method

[0076] The genomic DNA of a single corn plant is extracted as a template, and the KASP reaction is performed on all sample DNAs using the KASP primer set designed above. The entire KASP test process is performed using the SNPline genotyping platform and the data acquisition thereof. The PCR amplification reaction is performed on a high-throughput water bath (LGC company), and after the water bath reaches the temperature, the PCR plate is inserted vertically into the basket. The touch down PCR amplification program is as follows:

[0077] Stage 1, 95°C pre-denaturation for 15 min; stage 2, 94°C denaturation for 20 s, 61-55°C annealing for 1 min (decrease by 0.6°C for each cycle, 10 cycles), stage 3, 94°C denaturation for 20 s, 55°C annealing for 1 min (32 cycles).

[0078] The obtained PCR amplification product is subjected to genotyping on a PHERAstar plus fluorescent enzyme label instrument under fluorescence irradiation, and then the data after genotyping are read by KlusterCaller TM software.

[0079] According to the genotyping results, the mite resistance phenotype of the corn to be tested is determined as follows:

[0080] The leaf mite resistance of corn with the genotype GG of the SNP Zm00001d042333@161,630,118 site is greater than or candidate greater than that of corn with the genotype AA or AG of the SNP Zm00001d042333@161,630,118 site.

[0081] III. Application of SNP site Zm000011d042333@161,630,118

[0082] 1. Mite resistance phenotype identification

[0083] RIL007 x RIL009 two inbred lines were sowed in Hainan, and F1 seeds were obtained by hybridization. About 150 F1 seeds were sowed in Beijing Tongzhou in May 2023 to obtain F2 seeds about 130 ears, and about 1500 F2 plants were used for mite resistance phenotype identification.

[0084] The leaf mite grade results of 200 F2 plants are shown in Table 3.

[0085] Table 3 is the leaf mite grade of 200 F2 plants and the corresponding SNP genotype detected

[0086]

[0087]

[0088]

[0089] 2. SNP site identification

[0090] The primers of KASP molecular marker Zm00001d042333 (ZM-333) in the above-mentioned method 2 were used for KASP detection of the F2 population constructed by RIL007 x RIL009.

[0091] The KASP marker genotyping results are shown in Figure 3 A, and it can be seen that obvious genotyping is visible. Among them, G:G has 160, A:G has 409, and A:A has 183.

[0092] 3. SNP site identification and correlation with leaf mite resistance grade

[0093] The KASP marker genotyping of each sample in the above-mentioned 2 is plotted with its leaf mite grade.

[0094] The results are shown in Figure 3 B, and it can be seen that for KASP molecular marker Zm00001d042333 (ZM-333), the leaf mite resistance grade of AA or AG is greater than that of GG; it is shown that the leaf mite resistance of corn with SNP Zm00001d042333@161,630,118 site genotype GG is greater than that of corn with SNP Zm00001d042333@161,630,118 site genotype AA or AG.

[0095] The above results show that the leaf mite resistance of the corn to be tested can be detected or assisted by identifying the genotype of SNP Zm00001d042333@161,630,118 site, specifically as follows:

[0096] The genotype of SNP Zm00001d042333@161,630,118 site is detected, and the leaf mite resistance of the corn with the genotype of SNP Zm00001d042333@161,630,118 site being GG is greater than or candidate greater than the corn with the genotype of SNP Zm00001d042333@161,630,118 site being AA or AG.

[0097] Example 2, Application of SNP marker of corn mite resistance related gene

[0098] 1. Mite resistance phenotype identification

[0099] The samples of RIL284 (resistant, leaf mite level is secondary) and RIL004 (sensitive, leaf mite level is eight) were selected from the population of J92xJ2416 after mite resistance phenotype identification, and then F2 population of 1500 strains was constructed by crossing.

[0100] The F2 population constructed by RIL004xRIL284 was subjected to mite resistance phenotype identification in Tongzhou, Beijing in May 2023.

[0101] The leaf mite levels of 200 F2 plants and the corresponding detected SNP genotypes are as shown in Table 4.

[0102] Table 4 is the leaf mite level of 200 F2 plants and the corresponding detected SNP genotype

[0103]

[0104]

[0105] 2. SNP site identification

[0106] The primers of KASP molecular marker Zm00001d042333 (ZM-333) in the above-mentioned method two were used for KASP detection of the F2 population constructed by RIL004xRIL284.

[0107] The KASP marker genotyping results are as follows Figure 4 A, it can be seen that obvious genotyping can be seen. Among them, G: G has 284, A: G has 656, A: A has 285.

[0108] 3. SNP site identification and association with leaf mite resistance level

[0109] The KASP marker genotyping of each sample in the above 2 was plotted against its spider mite rating.

[0110] Results are shown in Table 2. Figure 4 B. It can be seen that for KASP molecular marker Zm00001d042333 (ZM-333), the spider mite resistance rating for AA or AG is greater than GG; indicating that corn with SNP Zm00001d042333@161,630,118 site genotype GG has greater spider mite resistance than corn with SNP Zm00001d042333@161,630,118 site genotype AA or AG.

Claims

1. Use of a primer for detecting the genotype of SNP site Zm000011d042333@161,630,118 in a corn genome in at least one of the following: A1) identifying or assisting in identifying corn resistance to spider mites; A2) breeding corn with high resistance to spider mites; wherein wherein the SNP site Zm000011d042333@161,630,118 is at position 26 of SEQ ID NO: 1, and the genotype of the SNP site Zm000011d042333@161,630,118 is GG, AA or AG, and wherein corn with the genotype GG at the SNP site Zm00001d042333@161,630,118 has greater resistance to spider mites than corn with the genotype AA or AG at the SNP site Zm00001d042333@161,630,118.

2. A primer set for detecting the genotype of SNP site Zm000011d042333@161,630,118 in a corn genome, or a PCR reagent or kit containing the primer set, the primer set comprising primer 1, primer 2 and primer 3; the nucleotide sequence of the primer 1 is shown in SEQ ID NO: 2; the nucleotide sequence of the primer 2 is shown in SEQ ID NO: 3; and the nucleotide sequence of the primer 3 is shown in SEQ ID NO: 4, wherein wherein the SNP site Zm000011d042333@161,630,118 is at position 26 of SEQ ID NO: 1, and the genotype of the SNP site Zm000011d042333@161,630,118 is GG, AA or AG, and wherein corn with the genotype GG at the SNP site Zm00001d042333@161,630,118 has greater resistance to spider mites than corn with the genotype AA or AG at the SNP site Zm00001d042333@161,630,118.

3. Use of the primer set for detecting the genotype of SNP site Zm000011d042333@161,630,118 in a corn genome of claim 2, or a PCR reagent or kit containing the primer set in at least one of the following: A1) identifying or assisting in identifying corn resistance to spider mites; A2) breeding corn with high resistance to spider mites; wherein the SNP site Zm000011d042333@161,630,118 is at position 26 of SEQ ID NO: 1, and the genotype of the SNP site Zm000011d042333@161,630,118 is GG, AA or AG, and wherein corn with the genotype GG at the SNP site Zm00001d042333@161,630,118 has greater resistance to spider mites than corn with the genotype AA or AG at the SNP site Zm00001d042333@161,630,118.

4. A method for identifying or assisting in identifying corn resistance to spider mites, comprising the following steps: detecting the genotype of the SNP site Zm000011d042333@161,630,118 in the corn genome as claimed in claim 1, and judging as follows: Corn with the genotype GG of the SNP site Zm00001d042333@161,630,118 has greater or is a candidate for greater resistance to spider mites than corn with the genotype AA or AG of the SNP site Zm00001d042333@161,630,118.

5. The method of claim 4, wherein: the detection of the genotype of the SNP site Zm000011d042333@161,630,118 in the corn genome as claimed in claim 1 is a KASP reaction of the corn genome using the kit of primers as claimed in claim 2, and the PCR amplification product obtained is genotyped.

6. A method for breeding corn with high resistance to spider mites, comprising the following steps: selecting corn with the genotype GG of the SNP site Zm00001d042333@161,630,118 in the method of claim 4 or 5 for breeding, and obtaining the desired corn. ​

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