Corn leafminer resistance-related molecular marker and application thereof

Abstract

The application discloses a corn spider mite resistance related molecular marker and application thereof. The application provides application of a substance for detecting the genotype of a SNP site Zm000011d042337@161,938,641 in a corn genome in at least one of the following: A1) identifying or assisting in identifying corn spider mite resistance; A2) breeding corn with high spider mite resistance; the SNP site Zm000011d042337@161,938,641 is the 23rd site in sequence 2. The application firstly finds two genes Zm000011d042333 and Zm000011d042337 related to mite resistance in corn, and identifies the mite resistance of corn according to the genotype of the SNP site on the gene, which creates a basis for breeding or screening resistant corn breeding resources.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to molecular markers related to spider mite resistance in maize and their applications. Background Technology

[0002] In actual production, maize is highly susceptible to pests and diseases, leading to large-scale yield reductions. However, the defense response of maize spider mites is still unclear, and research on its molecular mechanisms remains lacking. Screening for mite-resistant germplasm, especially for mite-resistant sources from commonly used maize backbone inbred lines in my country, is of great significance for the creation of new maize germplasm and sustainable agricultural development. Molecular markers can simultaneously identify multiple inbred lines. For the classification of a large number of inbred lines, molecular marker technology has shown significant advantages over other techniques. Developing mite-resistant molecular markers can be directly used for early identification and screening in routine genetic breeding, improving the breeding efficiency of new mite-resistant varieties.

[0003] Discovering mite resistance genes is crucial for breeding resistant varieties. The application of Kompetitive Allele Specific PCR (KASP) technology has identified multiple candidate genes for crop resistance and obtained functional molecular markers. Discovering spider mite resistance genes and developing related molecular markers has significant implications for the innovation of resistant maize germplasm and the breeding of new varieties.

[0004] Research on the genetic mechanisms of mite resistance in maize, especially in varieties native to my country, is very limited. Therefore, elucidating the genetic basis of mite resistance in maize is of great significance for the breeding and creation of mite-resistant varieties. Currently, the mechanisms of maize resistance to spider mites remain unclear, the genetic relationship between phenotype and genotype is not yet understood, and the associated molecular markers have not yet been discovered. There are currently no reports on molecular markers linking mite resistance phenotypic traits in maize leaves to spider mite severity ratings. Summary of the Invention

[0005] The purpose of this invention is to provide molecular markers for spider mite resistance-related genes in maize and their applications.

[0006] In a first aspect, the present invention provides a substance for detecting the genotype of the SNP locus Zm000011d042337@161,938,641 in the maize genome in at least one of the following applications:

[0007] A1) Identification or auxiliary identification of corn spider mite resistance;

[0008] A2) Breed maize with high resistance to spider mites;

[0009] The SNP site Zm000011d042337@161,938,641 is the 23rd position of sequence 2.

[0010] In the application described above, the genotype of the SNP site Zm000011d042337@161,938,641 is CC, TT, or TC.

[0011] In the application described above, the substance used to detect the SNP site Zm000011d042337@161,938,641 in the maize genome is 1) or 2):

[0012] 1) Primer set A;

[0013] 2) PCR reagents or kits containing the complete set of primers A;

[0014] The primer set includes primer 4, primer 5 and primer 6;

[0015] The nucleotide sequence of primer 4 includes the sequence shown in sequence 6;

[0016] The nucleotide sequence of primer 5 includes the sequence shown in sequence 7;

[0017] The nucleotide sequence of primer 6 is sequence 8.

[0018] Primer 4 mentioned above is an upstream primer with a fluorescent sequence added to the 5' end of the upstream primer shown in sequence 6; 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;

[0019] Primer 5, as described above, is an upstream primer with another fluorescent sequence added to the 5' end of the upstream primer shown in sequence 7; in an embodiment of the present invention, a 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.

[0020] Secondly, the present invention provides a substance for detecting the genotypes of SNP sites Zm000011d042333@161,630,118 and Zm000011d042337@161,938,641 in the maize genome, in at least one of the following applications:

[0021] A1) Identification or auxiliary identification of corn spider mite resistance;

[0022] A2) Breed maize with high resistance to spider mites;

[0023] The SNP site Zm000011d042333@161,630,118 is the 26th position of sequence 1;

[0024] The SNP site Zm000011d042337@161,938,641 is the 23rd position of sequence 2.

[0025] The genotype of the SNP locus Zm000011d042333@161,630,118 is GG, AA or AG;

[0026] The genotype of the SNP locus Zm000011d042337@161,938,641 is CC, TT, or TC.

[0027] In the application described above, the substance used to detect the SNP site Zm000011d042337@161,938,641 in the maize genome is 1) or 2):

[0028] 1) Primer set A;

[0029] 2) PCR reagents or kits containing the complete set of primers A;

[0030] The primer set includes primer 4, primer 5 and primer 6;

[0031] The nucleotide sequence of primer 4 includes the sequence shown in sequence 6;

[0032] The nucleotide sequence of primer 5 includes the sequence shown in sequence 7;

[0033] The nucleotide sequence of primer 6 is sequence 8;

[0034] The substance used to detect the SNP site Zm000011d042333@161,630,118 in the maize genome is 3) or 4):

[0035] 3) Primer set B;

[0036] 4) PCR reagents or kits containing the complete set of primers B;

[0037] The primer set B includes primer 1, primer 2 and primer 3;

[0038] The nucleotide sequence of primer 1 includes the sequence shown in sequence 3;

[0039] The nucleotide sequence of primer 2 includes the sequence shown in sequence 4;

[0040] The nucleotide sequence of primer 3 is sequence 5.

[0041] 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;

[0042] 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.

[0043] Thirdly, the present invention provides any of the following substances:

[0044] The primer set A described in the first aspect;

[0045] Alternatively, a PCR reagent or kit containing the complete set of primers A described in the first aspect;

[0046] Or the primer set A and primer set B described in the second aspect;

[0047] Alternatively, a PCR reagent or kit containing the primer set A and primer set B described in the second aspect.

[0048] Fourthly, the present invention provides for the use of the substances described in the third aspect in at least one of the following:

[0049] A1) Identification or auxiliary identification of corn spider mite resistance;

[0050] A2) Breed maize with high resistance to spider mites.

[0051] Fifthly, the present invention provides a method for identifying or assisting in the identification of maize spider mite resistance, comprising the following steps: detecting the genotype of the SNP site Zm000011d042337@161,938,641 described in the first aspect in the maize genome,

[0052] Maize with the genotype CC at the SNP Zm000011d042337@161,938,641 locus has greater or candidate greater resistance to spider mites than maize with the genotype TT or TC at the SNP Zm000011d042337@161,938,641 locus.

[0053] In the method described above, the genotype of the SNP site Zm000011d042337@161,938,641 in the first aspect of the maize genome is detected by performing a KASP reaction on the maize genome using the set of primers A described in the third aspect, and the PCR amplification product is then used for genotyping.

[0054] In a sixth aspect, the present invention provides a method for breeding maize with high resistance to spider mites, comprising the following steps: selecting maize with the genotype CC at the SNP Zm000011d042337@161,938,641 locus in the method described in the fifth aspect for breeding, thereby obtaining the target maize.

[0055] In the above method, the method for detecting whether the genotype of the Zm00001d042333@161,630,118 site in the maize genome to be tested is AA, GG, or AG is to perform PCR amplification on the maize genomic DNA to be tested using the above-mentioned set of primers, and then perform genotyping on the obtained PCR amplification products.

[0056] The method for detecting whether the genotype of the Zm000011d042337@161,938,641 site in the maize genome to be tested is CC, TT, or TC is to perform PCR amplification on the maize genomic DNA to be tested using the above-mentioned set of primers, and then perform genotyping on the obtained PCR amplification products.

[0057] In the above, the genotyping method after KASP detection of the Zm00001d042333@161,630,118 site is as follows: after irradiation with a fluorescent microplate reader, if the PCR product only shows the color of the fluorescent sequence attached to the 5' end of the DNA molecule as shown in sequence 3, then the genotype of the Zm00001d042333@161,630,118 site in the maize genome to be tested is G:G; if the PCR product only shows the color of the fluorescent sequence attached to the 5' end of the DNA molecule as shown in sequence 4, then the genotype of the Zm00001d042333@161,630,118 site in the maize genome to be tested is A:A.

[0058] If the PCR product shows the color of the fluorescent sequence attached to the 5' end of the DNA molecule shown in Sequence 3 and the color of the fluorescent sequence attached to the 5' end of the DNA molecule shown in Sequence 4, then the genotype of the Zm00001d042333@161,630,118 site in the maize genome to be tested is A:G.

[0059] In the above, the genotyping method after KASP detection of the Zm000011d042337@161,938,641 locus is as follows: after irradiation with a fluorescent microplate reader, if the PCR product only shows the color of the fluorescent sequence attached to the 5' end of the DNA molecule as shown in sequence 6, then the genotype of the Zm000011d042337@161,938,641 locus in the maize genome to be tested is C:C; if the PCR product only shows the color of the fluorescent sequence attached to the 5' end of the DNA molecule as shown in sequence 7, then the genotype of the Zm000011d042337@161,938,641 locus in the maize genome to be tested is T:T.

[0060] If the PCR product shows the color of the fluorescent sequence attached to the 5' end of the DNA molecule shown in Sequence 6 and the color of the fluorescent sequence attached to the 5' end of the DNA molecule shown in Sequence 7, then the genotype of the Zm000011d042337@161,938,641 site in the maize genome to be tested is T:C.

[0061] The corn mentioned above refers to the RIL populations of Jing 92 and Jing 2416 or their offspring. In this embodiment of the invention, the F2 generation of spider mite-resistant and spider mite-sensitive single plants from the RIL populations of Jing 92 and Jing 2416 is used; specifically, the F2 population of RIL007 (resistant, spider mite level 2) * RIL009 (sensitive, spider mite level 8) or the F2 population of RIL284 (resistant, spider mite level 2) * RIL004 (sensitive, spider mite level 8) from the RIL populations of Jing 92 and Jing 2416 is used.

[0062] This invention marks the first discovery of two genes in maize associated with mite resistance, Zm000011d042333 and Zm000011d042337. Furthermore, the genotype of the SNP loci on these genes was used to identify mite resistance in maize, providing a foundation for breeding or screening resistant maize resources. Based on these loci, a KASP marker was also designed. This marker can rapidly and accurately determine the genotype of the SNP locus and effectively screen resistant maize plants based on this genotype, thus accelerating maize resistance breeding. Attached Figure Description

[0063] Figure 1 Phenotypic identification of mite resistance in the population of Jing 92 (J92)*Jing 2416 (J2416). A: Image of maize leaf resistance to spider mites; B: Mite resistance rating in Hainan (21HN) in 2021 and Tongzhou, Beijing (22TZ) in 2022; C: Distribution of mite resistance rating of individual maize plants in leaves in Hainan in 2021 and Tongzhou, Beijing in 2022; D: Correlation between mite resistance rating of leaves in Hainan in 2021 and Tongzhou, Beijing in 2022.

[0064] Figure 2QTL mapping for the spider mite phenotype. A shows the mapping of the anti-mite trait on chromosome 3 in Hainan in 2021 and Tongzhou, Beijing in 2022; B shows all the preliminarily mapped QTL loci; C shows the intervals finely mapped based on 20 SNPs from last year.

[0065] Figure 3 The images show the KASP genotyping results of two candidate genes in the RIL007xRIL009 population and their corresponding spider mite resistance phenotypes. A shows the KASP genotyping results of the Zm00001d042333 gene; B shows the field spider mite resistance levels of different Zm00001d042333 genotypes; C shows the KASP genotyping results of the Zm00001d04237 gene; and D shows the field spider mite resistance levels of different Zm00001d042337 genotypes.

[0066] Figure 4 The images show the KASP genotyping results of two candidate genes in the RIL004xRIL284 population and their corresponding spider mite resistance phenotypes. A shows the KASP genotyping results of the Zm00001d042333 gene; B shows the field spider mite resistance levels of different Zm00001d042333 genotypes; C shows the KASP genotyping results of the Zm00001d04237 gene; and D shows the field spider mite resistance levels of different Zm00001d042337 genotypes. Detailed Implementation

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0068] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0069] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0070] In the following examples, the KASP reagent comprises three parts: sample DNA, KASP, and KASP. TM Components: primers and PCR system. Sample DNA was obtained via SDS-PAGE and KASP. TMThe Components primers consist of two forward primers and one reverse primer as the KASP Assay mix. The KASP Master mix contains a pair of FRET cassettes, ROX internal control dye, KlearTaq DNA polymerase, dNTPs, buffer, and MgCl2. For FRET structures in the PCR reaction system, the fluorophore shows no fluorescence signal before the reaction; during the reaction, the fluorophore binds to the corresponding sequence in two steps to generate fluorescence. The maize genome size is 2192.4 Mb, and the initial DNA concentration for the KASP experiment was 10 ng / μl. The final concentration for this experiment was 25 μmol / L.

[0071] KASP amplification program: Two-step landing PCR program at 61-55℃. Stage 1: Pre-denaturation at 95℃ for 15 min; Stage 2: Denaturation at 94℃ for 20 s, annealing at 61-55℃ for 1 min (decreasing by 0.6℃ per cycle, 10 cycles); Stage 3: Denaturation at 94℃ for 20 s, annealing at 55℃ for 1 min (32 cycles).

[0072] Genotyping data interpretation: Each data point represents a DNA sample. Samples with the same genotyping result are grouped together. Genotyping results are distinguished using the x-axis, y-axis, and diagonal of the coordinate system. Data closer to the y-axis is marked in red to indicate homozygous HEX marker alleles, data closer to the x-axis is marked in blue to indicate homozygous FAM marker alleles, and data on the diagonal is marked in green to indicate heterozygous FAM marker alleles and HEX marker alleles.

[0073] The example below is described in the following literature: Zhao Jiuran et al., Breeding and application of maize backbone inbred line Jing 2416, Journal of Plant Genetic Resources, 2020.3.12;

[0074] Jing 92 is recorded in the following literature: Chen Yafeng et al., Effects of leaf physical traits on the defense of maize hybrid Jingke 968 against two-spotted spider mite, Journal of Environmental Entomology, 2022, 44(1):229-235.

[0075] Example 1: Obtaining SNP markers for genes related to mite resistance in maize

[0076] I. Discovery of SNP sites of genes related to mite resistance in maize

[0077] Using Jing 2416 as the female parent and Jing 92 as the parent, hybridization was carried out to obtain F1. Then, F1 was self-crossed to F2. After F2, one grain was selected from each ear and planted for self-crossing. This process was repeated until F6 was obtained, which is the RIL population (this population is named the J92*J2416 population).

[0078] Using 318 individuals from the population of Jing 92 (J92) * Jing 2416 (J2416), at two locations, Tongzhou, Beijing in May 2022 and Hainan in October 2021 respectively, they were grown until the tasseling stage, and the degree of damage to maize leaves after infestation was investigated to conduct phenotypic identification of mite resistance (for the method, see: Di et al, 2014, Laboratory and field evaluation of maize resistance to the two-spotted spider mite, Tetranychus urticae).

[0079] The investigation criteria for spider mite feeding on leaves are as follows. The grading criteria are shown in Table 1 below: When the ear leaf is eaten by insects, it is defined as a third-level hazard, and when it is severe, it is defined as a fourth-level hazard; for each additional leaf above the ear leaf, the hazard is defined as one level higher (for example, the leaf above the ear leaf is often defined as a fourth-level hazard, and the two leaves above the ear leaf are often defined as a fifth-level hazard). Generally, a score of 1 - 3 indicates strong mite resistance, and a score of 8 - 10 indicates mite sensitivity. The higher the level, the poorer the mite resistance.

[0080] Table 1 is the grading standard for spider mite levels

[0081]

[0082] The two phenotypic data were significantly correlated. The mite resistance phenotype showed an additive effect, and both phenotypes conformed to a normal distribution ( Figure 1 ).

[0083] QTL mapping was performed on the population mite resistance phenotypes of the above two populations of planting materials J92 * J2416 ( Figure 2 ). The results showed that a 15M region on chromosome 3 was commonly mapped in both cases. The phenotypic contribution rates were 12.1% in Hainan in 2021 and 4.3% in Tongzhou in 2022, respectively, and it contains 237 genes. It is a new QTL locus derived from J92, different from the B96 resistance source of the American line (chromosome 6).

[0084] After identifying the anti-mite phenotype in the J92*J2416 population, samples RIL007 (resistant, spider mite grade 2) and RIL009 (sensitive, spider mite grade 8) were selected for hybridization to construct an F2 population of 1468 plants. Twenty new markers were developed within a 15M region for fine mapping of anti-mite loci, and candidate genes were screened. Two non-synonymous SNP loci were anchored on the candidate genes Zm000011d042333 and Zm000011d042337. The two loci were determined by comparing the gene sequences of J92 and J2416. The two nonsynonymous SNPs are located at positions 161,630,118 and 161,938,641 in the B73 reference genome (Zm-B73-REFERENCE-GRAMENE-4.0 February 2017). In anti-mite studies, they involve mutating nonsynonymous G to A and C to T.

[0085] Therefore, the two non-synonymous SNPs are named Zm000011d042333@161,630,118 and Zm000011d042337@161,938,641, respectively.

[0086] Zm000011d042333@161,630,118 is located at position 161,630,118 of the maize B73 reference genome or at position 26 of sequence 1. The genotype of this SNP site is GG, AA, or AG; Sequence 1: TGCTTTCAGATTCCTGCGAGCAATGRTGAATGCATCTATTCT, where R is G or A.

[0087] Zm000011d042337@161,938,641 is located at position 161,938,641 of the maize B73 reference genome or at position 23 of sequence 2. The genotype of this SNP site is CC, TT, or TC. Sequence 2: TACAAAACCACCCTTCTGATTCYRATAGTTGYCATGACGCATGCAGGACGTG, where Y is C or T and R is A or G.

[0088] II. Establishment of a method for detecting SNPs Zm000011d042333@161,630,118 and Zm000011d042337@161,938,641

[0089] 1. Design and synthesis of KASP primers

[0090] To further verify the relationship between the SNP site and the leaf-feeding phenotypic phenotype of spider mites, KASP analysis was performed on the potential functional SNP in the entire localized population. Primers were designed as follows:

[0091] 1) Primers for the KASP molecular marker Zm00001d042333 (ZM-333)

[0092] Based on the nucleotide sequences before and after SNP Zm00001d042333@161,630,118 in sequence 1, a set of KASP primers (denoted as KASP molecular marker Zm00001d042333 (ZM-333)) was designed to determine the SNP Zm00001d042333@161,630,118 site as follows:

[0093] Upstream primer: F5'-CTGAAACTATAAGAATAGATGCATTCAC-3' (sequence 3),

[0094] Upstream primer: F5'-GTCTGAAACTATAAGAATAGATGCATTCAT-3' (sequence 4),

[0095] Downstream primer: R5'-CTAATGCTTTCAGATTCCTGCGAGCAA-3' (sequence 5).

[0096] Add the specific fluorescent sequence FAM: 5'-GAAGGTGACCAAGTTCATGCT-3' to the 5' end of the upstream primer shown in Sequence 3 to obtain an upstream primer with the specific fluorescent sequence FAM added to the 5' end;

[0097] Add the specific fluorescent sequence HEX: F5'-GAAGGTCGGAGTCAACGGATT-3' to the 5' end of the upstream primer shown in Sequence 4 to obtain an upstream primer with the specific fluorescent sequence HEX added to the 5' end.

[0098] The upstream primer with the specific fluorescent sequence FAM added to the 5' end (after adding FAM to sequence 3) amplifies the fragment with genotype A:A at the SNP Zm00001d042333@161,630,118 site using the single-stranded DNA molecule shown in sequence 5. The product of PCR amplification carrying the FAM sequence appears blue when irradiated with fluorescence.

[0099] The upstream primer with the specific fluorescent sequence HEX added to the 5' end (after adding HEX to sequence 4) amplifies the fragment with genotype G:G at the SNP Zm00001d042333@161,630,118 site using the single-stranded DNA molecule shown in sequence 5. The PCR product carrying the HEX sequence shows a red color when irradiated with fluorescence.

[0100] The upstream primers with the specific fluorescent sequence FAM added to the 5' end, the upstream primers with the specific fluorescent sequence HEX added to the 5' end, and the single-stranded DNA molecule shown in Sequence 5 amplified the fragment with genotype A:G at the SNP Zm00001d042333@161,630,118 locus. The PCR amplification product showed green color when illuminated by fluorescence.

[0101] Table 2 shows the primer design, upstream and downstream sequences, and base mutation patterns for the KASP molecular marker Zm00001d042333 (ZM-333). The 142nd position in the table is the SNP site.

[0102] Table 2 shows the information on KASP molecular markers.

[0103]

[0104] 2) Primers for the KASP molecular marker Zm00001d042337 (ZM-337)

[0105] Based on the nucleotide sequences before and after sequence 2 of SNP Zm000011d042337@161,938,641, a set of KASP primers (denoted as KASP molecular marker Zm00001d042337 (ZM-337)) was designed to determine the SNP Zm000011d042337@161,938,641 site as follows:

[0106] Upstream primer: F5'-TTACAAAACCACCCTTCTGATTCC-3' (sequence 6),

[0107] Upstream primer: F5'-CTTTACAAAACCACCCTTCTGATTCT-3' (sequence 7),

[0108] Downstream primer: R5'-GTCCTGCATGCGTCATGRCAACTA-3' (sequence 8), where R is either A or G.

[0109] Add the specific fluorescent sequence FAM: 5'-GAAGGTGACCAAGTTCATGCT-3' to the 5' end of the upstream primer shown in Sequence 6 to obtain an upstream primer with the specific fluorescent sequence FAM added to the 5' end;

[0110] Add the specific fluorescent sequence HEX: F5'-GAAGGTCGGAGTCAACGGATT-3' to the 5' end of the upstream primer shown in Sequence 7 to obtain an upstream primer with the specific fluorescent sequence HEX added to the 5' end.

[0111] The upstream primer with the specific fluorescent sequence FAM added to the 5' end (after adding FAM to sequence 6) amplifies the fragment with genotype T:T at the SNP Zm000011d042337@161,938,641 site using the single-stranded DNA molecule shown in sequence 8. The product of PCR amplification carrying the FAM sequence appears blue when irradiated with fluorescence.

[0112] The upstream primer with the specific fluorescent sequence HEX added to the 5' end (after adding HEX to sequence 7) amplifies the C:C fragment of the SNP Zm000011d042337@161,938,641 locus by the single-stranded DNA molecule shown in sequence 8. The PCR product carrying the HEX sequence shows red fluorescence when irradiated.

[0113] The upstream primers with the specific fluorescent sequence FAM added to the 5' end, the upstream primers with the specific fluorescent sequence HEX added to the 5' end, and the single-stranded DNA molecule shown in Sequence 8 amplified the fragment with the genotype T:C at the SNP Zm000011d042337@161,938,641 locus. The PCR amplification product showed green color when illuminated by fluorescence.

[0114] The primer design, upstream and downstream sequences, and base mutation patterns for the KASP molecular marker Zm00001d042337 (ZM-337) are shown in Table 3. The 186th position of the sequence in the table is the SNP site.

[0115] Table 3 shows the information on KASP molecular markers.

[0116]

[0117] 2. KASP reaction method

[0118] Genomic DNA was extracted from individual maize plants as templates, and KASP reactions were performed on all sample DNA using the KASP primer sets corresponding to each SNP locus designed above. The entire KASP experimental procedure utilized the SNPline genotyping platform for experimentation and data acquisition. PCR amplification reactions were performed in a high-throughput water bath (LGC). After the water bath reached the desired temperature, the PCR plate was vertically inserted into the basket. The touch-down PCR amplification program was used as follows:

[0119] Stage 1: Pre-degradation at 95℃ for 15 min; Stage 2: Degradation at 94℃ for 20 s, annealing at 61-55℃ for 1 min (decreasing by 0.6℃ per cycle, 10 cycles); Stage 3: Degradation at 94℃ for 20 s, annealing at 55℃ for 1 min (32 cycles).

[0120] The obtained PCR amplification products were processed in PHERAstar. plusGenotyping was performed using a fluorescent microplate reader with fluorescence illumination, and then in KlusterCaller. TM The software reads the data after fracturing.

[0121] Based on the typing results, determine the mite resistance phenotype of the maize sample according to any of the following methods:

[0122] 1) Maize with the genotype GG at the SNP Zm00001d042333@161,630,118 locus has greater resistance to spider mites than maize with the genotype AA or AG at the SNP Zm00001d042333@161,630,118 locus.

[0123] 2) Maize with the genotype CC at the SNP Zm000011d042337@161,938,641 locus has greater or candidate greater resistance to spider mites than maize with the genotype TT or TC at the SNP Zm000011d042337@161,938,641 locus.

[0124] 3) Maize with genotype GG at SNP Zm00001d042333@161,630,118 and genotype CC at SNP Zm000011d042337@161,938,641 has greater or candidate greater resistance to spider mites than maize with genotype AA or AG at SNP Zm00001d042333@161,630,118, or genotype TT or TC at SNP Zm000011d042337@161,938,641.

[0125] III. Application of SNP site Zm000011d042333@161,630,118

[0126] 1. Identification of anti-mite phenotypes

[0127] Two inbred lines, RIL007 and RIL009, were sown in Hainan and hybridized to obtain F1 seeds. Approximately 150 F1 seeds were sown in Tongzhou, Beijing in May 2023 and self-pollinated to obtain approximately 130 F2 ears. Approximately 1,800 F2 plants were then subjected to mite resistance phenotype identification.

[0128] For example, the partial results of the spider mite severity of 200 F2 plants are shown in Table 4 below.

[0129] Table 4 shows the spider mite severity levels and corresponding SNP genotypes of 200 F2 plants.

[0130]

[0131]

[0132]

[0133] 2. SNP locus identification

[0134] The primers for the KASP molecular marker Zm00001d042333 (ZM-333) from the method described above were used to perform KASP detection on the F2 population constructed from RIL007xRIL009.

[0135] KASP marker gene typing results are as follows Figure 3 As shown in Figure A, clear genotyping is evident. There are 160 G:G types, 409 A:G types, and 183 A:A types.

[0136] 3. Identification of SNP sites and their correlation with spider mite resistance levels

[0137] Plot the KASP marker gene typing of each sample in the above 2 samples and their corresponding spider mite grades.

[0138] The results are as follows Figure 3 As shown in Figure B, it can be seen that for the KASP molecular marker Zm00001d042333 (ZM-333), the spider mite resistance level of AA or AG is greater than that of GG; indicating that maize with the genotype GG at the SNP Zm00001d042333@161,630,118 site has greater spider mite resistance than maize with the genotype AA or AG at the SNP Zm00001d042333@161,630,118 site.

[0139] IV. Application of SNP site Zm000011d042337@161,938,641

[0140] 1. Identification of anti-mite phenotypes

[0141] Two inbred lines, RIL007 and RIL009, were sown in Hainan and hybridized to obtain F1 seeds. Approximately 150 F1 seeds were sown in Tongzhou, Beijing in May 2023 and self-pollinated to obtain approximately 130 F2 ears. Approximately 1,800 F2 plants were then subjected to mite resistance phenotype identification.

[0142] Table 5 below shows the results of spider mite severity and corresponding SNP genotype detection for 200 F2 plants.

[0143] Table 5 shows the spider mite severity and corresponding SNP genotypes of 200 F2 plants.

[0144]

[0145]

[0146]

[0147] 2. SNP locus identification

[0148] The primers for the KASP molecular marker Zm00001d042337 (ZM-337) from the method described above were used to perform KASP detection on the F2 population constructed from RIL007xRIL009.

[0149] KASP marker genotyping results are as follows: Figure 3 As shown in Figure C, clear genotyping is evident. There are 167 C:C pairs, 180 T:T pairs, and 393 T:C pairs.

[0150] 3. Identification of SNP sites and their correlation with spider mite resistance levels

[0151] Plot the KASP marker gene typing of each sample in the above 2 samples and their corresponding spider mite grades.

[0152] The results are as follows Figure 3 As shown in Figure D, it can be seen that for the KASP molecular marker Zm00001d042337 (ZM-337), the spider mite resistance level of TT or TC is greater than that of CC; indicating that maize with the genotype CC at the SNP Zm000011d042337@161,938,641 site has greater spider mite resistance than maize with the genotype TT or TC at the SNP Zm000011d042337@161,938,641 site.

[0153] The above results indicate that the spider mite resistance of maize samples can be detected or assisted in by identifying the genotypes of SNP Zm00001d042333@161,630,118 or SNP Zm000011d042337@161,938,641 loci, as detailed below:

[0154] Genotypes at SNP Zm00001d042333@161,630,118 were detected. Maize with the genotype GG at SNP Zm00001d042333@161,630,118 showed greater or candidate greater resistance to spider mites than maize with the genotype AA or AG at SNP Zm00001d042333@161,630,118.

[0155] Alternatively, the genotype of SNP Zm000011d042337@161,938,641 can be detected. Maize with the genotype CC at SNP Zm000011d042337@161,938,641 has greater or candidate greater spider mite resistance than maize with the genotype TT or TC at SNP Zm000011d042337@161,938,641.

[0156] Alternatively, the genotypes of two SNP loci can be used together to determine the resistance. Specifically, the genotypes of SNP Zm00001d042333@161,630,118 and SNP Zm000011d042337@161,938,641 can be detected. Maize with a genotype of GG at SNP Zm00001d042333@161,630,118 and a genotype of CC at SNP Zm000011d042337@161,938,641 has a greater or candidate greater resistance to spider mites than maize with a genotype of AA or AG at SNP Zm00001d042333@161,630,118 or a genotype of TT or TC at SNP Zm000011d042337@161,938,641.

[0157] Example 2: Application of SNP markers for genes related to mite resistance in maize

[0158] I. Application of SNP site Zm000011d042333@161,630,118

[0159] 1. Identification of anti-mite phenotypes

[0160] After identifying the anti-mite phenotype in the J92*J2416 population, samples RIL284 (resistant, spider mite grade 2) and RIL004 (sensitive, spider mite grade 8) were selected and crossbred to construct an F2 population of 1800 plants.

[0161] The F2 population constructed from RIL004xRIL284 was subjected to anti-mite phenotype identification in Tongzhou, Beijing in May 2023.

[0162] Table 6 below shows the spider mite severity levels and corresponding SNP genotypes of 200 F2 plants.

[0163] Table 6 shows the spider mite severity and corresponding SNP genotypes of 200 F2 plants.

[0164]

[0165]

[0166]

[0167] 2. SNP locus identification

[0168] The primers for the KASP molecular marker Zm00001d042333 (ZM-333) from the method described above were used to perform KASP detection on the F2 population constructed from RIL004xRIL284.

[0169] KASP marker genotyping results are as follows: Figure 4 As can be seen from A, there are obvious genotypes. Among them, there are 284 G:G types, 656 A:G ​​types, and 285 A:A types.

[0170] 3. Identification of SNP sites and their correlation with spider mite resistance levels

[0171] Plot the KASP marker gene typing of each sample in the above 2 samples and their corresponding spider mite grades.

[0172] The results are as follows Figure 4 B. It can be seen that, for the KASP molecular marker Zm00001d042333 (ZM-333), the spider mite resistance level of AA or AG is greater than that of GG; indicating that maize with the genotype GG at the SNP Zm00001d042333@161,630,118 site has greater spider mite resistance than maize with the genotype AA or AG at the SNP Zm00001d042333@161,630,118 site.

[0173] II. Application of SNP site Zm000011d042337@161,938,641

[0174] 1. Identification of anti-mite phenotypes

[0175] After identifying the anti-mite phenotype in the J92*J2416 population, samples RIL284 (resistant, spider mite grade 2) and RIL004 (sensitive, spider mite grade 8) were selected and crossbred to construct an F2 population of 1800 plants.

[0176] Two inbred lines, RIL004 and RIL284, were sown in Hainan and hybridized to obtain F1 seeds. Approximately 150 F1 seeds were sown in Tongzhou, Beijing in May 2023 and self-pollinated to obtain approximately 130 F2 ears. About 1,800 F2 plants were then subjected to phenotypic identification.

[0177] Table 7 below shows the spider mite severity levels and corresponding SNP genotypes of 200 F2 plants.

[0178] Table 7 shows the spider mite severity and corresponding SNP genotypes of 200 F2 plants.

[0179]

[0180]

[0181] 2. SNP locus identification

[0182] The primers for the KASP molecular marker Zm00001d042337 (ZM-337) from the method described above were used to perform KASP detection on the F2 population constructed from RIL004xRIL284.

[0183] KASP marker genotyping results are as follows: Figure 4 As can be seen from C, there are obvious genotyping patterns. Among them, there are 241 C:C pairs, 230 T:T pairs, and 497 C:T pairs.

[0184] 3. Identification of SNP sites and their correlation with spider mite resistance levels

[0185] Plot the KASP marker gene typing of each sample in the above 2 samples and their corresponding spider mite grades.

[0186] The results are as follows Figure 4 D. It can be seen that, for the KASP molecular marker Zm00001d042337 (ZM-337), the spider mite resistance level of TT or TC is greater than that of CC; indicating that maize with genotype CC at SNP Zm000011d042337@161,938,641 has greater spider mite resistance than maize with genotype TT or TC at SNP Zm000011d042337@161,938,641.

Claims

1. The substance used to detect the genotype of the SNP locus Zm000011d042337@161,938,641 in the maize genome has at least one of the following applications: A1) Identification or auxiliary identification of corn spider mite resistance; A2) Breed maize with high resistance to spider mites; The SNP site Zm000011d042337@161,938,641 is the 23rd position of sequence 2, and the genotype of the SNP site Zm000011d042337@161,938,641 is CC, TT or TC.

2. The application according to claim 1, characterized in that: The substance used to detect the SNP site Zm000011d042337@161,938,641 in the maize genome is 1) or 2): 1) Primer set A; 2) PCR reagents or kits containing the complete set of primers A; The primer set A includes primer 4, primer 5 and primer 6; The nucleotide sequence of primer 4 includes the sequence shown in sequence 6; The nucleotide sequence of primer 5 includes the sequence shown in sequence 7; The nucleotide sequence of primer 6 is sequence 8.

3. The substance used to detect the genotypes of SNP sites Zm000011d042333@161,630,118 and Zm000011d042337@161,938,641 in the maize genome has at least one of the following applications: A1) Identification or auxiliary identification of corn spider mite resistance; A2) Breed maize with high resistance to spider mites; The SNP site Zm000011d042333@161,630,118 is the 26th position of sequence 1, and the genotype of the SNP site Zm000011d042333@161,630,118 is GG, AA or AG; The SNP site Zm000011d042337@161,938,641 is the 23rd position of sequence 2, and the genotype of the SNP site Zm000011d042337@161,938,641 is CC, TT or TC.

4. The application according to claim 3, characterized in that: The substance used to detect the SNP site Zm000011d042337@161,938,641 in the maize genome is 1) or 2): 1) Primer set A; 2) PCR reagents or kits containing the complete set of primers A; The primer set A includes primer 4, primer 5 and primer 6; The nucleotide sequence of primer 4 includes the sequence shown in sequence 6; The nucleotide sequence of primer 5 includes the sequence shown in sequence 7; The nucleotide sequence of primer 6 is sequence 8; The substance used to detect the SNP site Zm000011d042333@161,630,118 in the maize genome is 3) or 4): 3) Primer set B; 4) PCR reagents or kits containing the complete set of primers B; The primer set B includes primer 1, primer 2 and primer 3; The nucleotide sequence of primer 1 includes the sequence shown in sequence 3; The nucleotide sequence of primer 2 includes the sequence shown in sequence 4; The nucleotide sequence of primer 3 is sequence 5.

5. Any of the following substances: The primer set A as described in claim 2; Alternatively, a PCR reagent or kit containing the complete set of primers A as described in claim 2; Or the primer set A and primer set B as described in claim 4; Alternatively, a PCR reagent or kit containing the primer set A and primer set B as described in claim 4.

6. The use of each of the substances described in claim 5 in at least one of the following: A1) Identification or auxiliary identification of corn spider mite resistance; A2) Breed maize with high resistance to spider mites.

7. A method for identifying or assisting in the identification of maize spider mite resistance, comprising the following steps: detecting the genotype of the SNP locus Zm000011d042337@161,938,641 in the maize genome as described in claim 1, Maize with the genotype CC at the SNP Zm000011d042337@161,938,641 locus has greater or candidate greater resistance to spider mites than maize with the genotype TT or TC at the SNP Zm000011d042337@161,938,641 locus.

8. The method according to claim 7, characterized in that: The method for detecting the genotype of the SNP site Zm000011d042337@161,938,641 in the maize genome according to claim 1 is to perform a KASP reaction on the maize genome using the set of primers A described in claim 5, and then perform genotyping on the PCR amplification products obtained.

9. A method for breeding maize with high resistance to spider mites, comprising the following steps: selecting maize with the genotype CC at the SNP Zm000011d042337@161,938,641 locus in the method of claim 7 or 8 for breeding to obtain the target maize.

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