Application of ZmSMR1 in Maize Germplasm Creation and Variety Breeding

Abstract

This invention provides ZmSMR1, ZmSMR2 and ZmSMR3 Its application in maize germplasm creation and variety breeding, especially in maize breeding, and the provision of methods to improve the resistance of maize to spider mites and to breed highly spider mite-resistant maize, have significantly accelerated the forward and backcross breeding process of new highly spider mite-resistant maize varieties.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to... ZmSMR1 , ZmSMR2 and ZmSMR3 Application in maize germplasm creation and variety breeding. Background Technology

[0002] Two-spotted spider mite ( Tetranychus urticae Tetranychus koch is a highly destructive phytophagous mite whose host range includes more than 1,100 plant species, including maize (Santamaria et al., 2020; Zhang et al., 2025). Tetranychus suck the contents of plant cells with their stylets, causing leaf chlorosis and necrosis. Due to their short lifespan, rapid development of acaricide resistance (Tudi et al., 2021), and preference for feeding on the undersides of leaves, chemical control is ineffective, posing a significant threat to global agricultural production (Xu et al., 2018; Assouguem et al., 2022). Hot, dry climates favor rapid population growth of Tetranychus (Santamaría et al., 2019; Ma et al., 2025); under drought stress in the United States, maize yield losses have reached as high as 47% (Maxmen, 2013). In China, spider mite infestation is escalating, becoming one of the most significant pests in the Northwest corn-producing region (Wang & Wang, 2019; Chen et al., 2020). In some areas, the number of spider mites per 100 corn plants has exceeded 160,000 (Center, 2013); official Chinese data shows that the affected area will reach 4.47 million hectares nationwide by 2024, and the damage is expected to worsen globally (Wang & Wang, 2019).

[0003] Since spider mites feed on leaves using their piercing-sucking mouthparts, the plant cell wall is the first physical barrier, and its thickness and mechanical properties directly affect the efficiency of stylet penetration (Cui et al., 2024; Hashemi et al., 2025). Constitutive features such as increased cuticle and cell wall thickness have been shown to be associated with resistance to spider mites in cotton, pigeon pea, and apple (Tutushkina N, 2025; Jiang et al., 2024); while induced defense responses strengthen cell wall structure through the accumulation of lignin and condensed tannins, thereby increasing resistance to spider mites in cassava and cucumber (Chen et al., 2022; Yao et al., 2022).

[0004] Despite these existing understandings, the genetic structure behind maize spider mite resistance and the role of cell wall remodeling in this process remain not fully elucidated. To date, due to the polygenic and quantitative nature of spider mite resistance, no maize mite resistance gene has been cloned or functionally identified (Rosa-Diaz et al., 2025).

[0005] The improved inbred line Jing 92 of the Chinese Huangzao 4 variety exhibits strong resistance to spider mites under both greenhouse and field conditions; however, the genetic basis of this resistance remains unclear. Jing 92 is the germplasm source for the two-spotted spider mite resistance of the maize variety Jingke 968. After infection, Jing 92 exhibits low total mite populations, low number of adult females, low hatching rate, and low reproductive capacity, resulting in an 89% reduction in field spider mite density (Di et al., 2024). This resistance may be related to leaf physical traits (such as smaller stomatal size and thicker leaves). However, the causal genes and molecular mechanisms are still unclear, hindering its application in breeding.

[0006] Therefore, identifying QTLs, candidate genes, and molecular markers is of great significance for applying marker-assisted selection in breeding practices, which will significantly accelerate the forward and backcross breeding process of new maize varieties with high resistance to spider mites. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide ZmSMR1 , ZmSMR2 and ZmSMR3 Its application in maize germplasm creation and variety breeding is aimed at cultivating maize varieties with excellent resistance to spider mites.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: In a first aspect, the present invention provides ZmSMR1 The application of genes in improving maize's resistance to spider mites, the ZmSMR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the application includes upregulating the gene. ZmSMR1 Gene expression in maize can enhance its resistance to spider mites.

[0009] Optionally, the present invention also provides ZmSMR1 The application of genes in genetic breeding to improve the resistance of maize to spider mites, germplasm resource improvement to improve the resistance of maize to spider mites, and in the preparation of transgenic maize crops resistant to spider mites. ZmSMR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the application includes upregulating the gene. ZmSMR1 Gene expression in maize can enhance its resistance to spider mites.

[0010] Optionally, the present invention also provides ZmSMR1 The application of genes in regulating maize cell wall structure, through upregulation ZmSMR1 Gene expression in maize increases the content of pectin and hemicellulose in maize cell walls, remodels cell wall structure, and thickens leaves and cell walls; ZmSMR1 The gene encodes the GT47 family of glycosyltransferases, which ZmSMR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

[0011] Optionally, the present invention also provides a breeding method for maize with high resistance to spider mites, including by upregulating... ZmSMR1 The expression of the gene in maize enhances the maize's resistance to spider mites, wherein... ZmSMR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

[0012] In a second aspect, the present invention provides ZmSMR2 Application of genes in improving maize's resistance to spider mites ZmSMR2 The nucleotide sequence of the gene is shown in SEQ ID NO. 3, and its applications include upregulation. ZmSMR2 Gene expression in maize can enhance its resistance to spider mites.

[0013] Optionally, the present invention also provides ZmSMR2 Application of genes in genetic breeding to improve the resistance of maize to spider mites. ZmSMR2 The nucleotide sequence of the gene is shown in SEQ ID NO. 3, and its applications include upregulation. ZmSMR2 Gene expression in maize can enhance its resistance to spider mites.

[0014] Optionally, the present invention also provides ZmSMR2 Application of genes in germplasm improvement to enhance the resistance of maize to spider mites. ZmSMR2 The nucleotide sequence of the gene is shown in SEQ ID NO. 3, and its applications include upregulation. ZmSMR2 Gene expression in maize can enhance its resistance to spider mites.

[0015] Optionally, the present invention also provides ZmSMR2 Application of genes in the production of transgenic maize resistant to spider mites ZmSMR2 The nucleotide sequence of the gene is shown in SEQ ID NO. 3, and its applications include upregulation. ZmSMR2 Gene expression in maize can enhance its resistance to spider mites.

[0016] Optionally, the present invention also provides a breeding method for maize with high resistance to spider mites, comprising the following steps: by upregulating ZmSMR2 The expression of genes in maize enhances its resistance to spider mites. ZmSMR2 The nucleotide sequence of the gene is shown in SEQ ID NO. 3.

[0017] In a third aspect, the present invention provides ZmSMR3 The application of the gene in improving the performance of maize against spider mites, characterized in that ZmSMR3 The nucleotide sequence of the gene is shown in SEQ ID NO. 5, and the application includes up-regulating ZmSMR3 the expression of the gene in maize to improve the resistance of maize to spider mites.

[0018] Optionally, the present invention also provides ZmSMR3 The application of the gene in genetic breeding for improving the performance of crop maize against spider mites, ZmSMR3 The nucleotide sequence of the gene is shown in SEQ ID NO. 5, and the application includes up-regulating ZmSMR3 the expression of the gene in maize to improve the resistance of maize to spider mites.

[0019] Optionally, the present invention also provides the application of the mSMR3 gene in improving the germplasm resources of crop maize against spider mites, ZmSMR3 The nucleotide sequence of the gene is shown in SEQ ID NO. 5, and the application includes up-regulating ZmSMR3 the expression of the gene in maize to improve the resistance of maize to spider mites.

[0020] Optionally, the present invention also provides ZmSMR3 The application of the gene in preparing transgenic crop maize resistant to spider mites, ZmSMR3 The nucleotide sequence of the gene is shown in SEQ ID NO. 5, and the application includes up-regulating ZmSMR3 the expression of the gene in maize to improve the resistance of maize to spider mites.

[0021] Optionally, the present invention also provides a breeding method for highly spider mite-resistant maize, comprising the following steps: by up-regulating ZmSMR3 the expression of the gene in maize to improve the resistance of maize to spider mites, wherein ZmSMR3 the nucleotide sequence of the gene is shown in SEQ ID NO. 5.

[0022] Compared with the prior art, the beneficial effects of the present invention are: The present invention uses a recombinant inbred line population derived from Jing 92×Jing 2416 to map a major QTL-qSMR3-1 related to leaf thickness, qSMR3-1 As a major QTL identified in Hainan, it covers a 15-Mb genomic region containing 237 genes. And it is finely mapped to a 1-Mb interval, and then the ZmSMR1 gene is cloned. The ZmSMR1The gene encodes a glycosyltransferase of the GT47 family. Functional verification confirmed that ZmSMR1 it positively regulates Tetranychus urticae resistance. In addition, ZmSMR1 it remodels the cell wall structure by regulating the levels of pectin and hemicellulose, which is related to the increase in the leaf and cell wall thickness of the resistant inbred line Jing 92. ZmSMR1 The knockout transgenic lines showed a significant increase in susceptibility after Tetranychus urticae infestation, and at the same time, the cell wall thickness and leaf thickness decreased significantly. Compared with Jing 92, the pectin and hemicellulose contents in the knockout lines decreased significantly, as did the rhamnose and galacturonic acid contents, indicating that ZmSMR1 it may mediate the change of leaf physical traits through cell wall remodeling, thereby regulating Tetranychus urticae resistance. Kompetitive allele-specific PCR analysis showed that ZmSMR1 the functional SNP in

[0023] The present invention provides ZmSMR1 the application of the ZmSMR1 gene in improving maize resistance to Tetranychus urticae, especially

[0024] in the creation of maize germplasm and variety breeding, and provides a method for improving the Tetranychus urticae resistance of the crop maize and a method for breeding maize highly resistant to Tetranychus urticae, which significantly accelerates the forward and backcross breeding processes of new maize varieties highly resistant to Tetranychus urticae.

[0024] The present invention uses a recombinant inbred line population derived from Jing 92 × Jing 2416 to map a major QTL related to Tetranychus urticae resistance and finely determine the candidate ZmSMR2 gene. ZmSMR2 The knockout transgenic lines showed a significant increase in susceptibility after Tetranychus urticae infestation. ZmSMR2 The functional SNP in

[0025] The present invention uses a recombinant inbred line population derived from Jing 92 × Jing 2416 to map a major QTL related to Tetranychus urticae resistance and finely determine the candidate ZmSMR3 gene. ZmSMR3 The knockout transgenic lines showed a significant increase in susceptibility after Tetranychus urticae infestation. [4]] ZmSMR3 The functional SNP in Brief Description of the Drawings

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art.

[0027] Figure 1 It is the phenotype of "Jing 92" and "Jing 2416" after mite infestation and the number of Tetranychus urticae per plant.

[0028] Figure 2 This is a characterization of the results of phenotypic analysis of spider mite damage levels and leaf thickness in 318 recurrent populations generated by the hybridization of "Jing 92" × "Jing 2416".

[0029] Figure 3 Candidate genes for resistance to spider mites in the Jing92 strain ( ZmSMR1 Cloning, expression levels, and subcellular localization analysis of ).

[0030] Figure 3a These are the carrier maps of PG0038 and PG0039; Figure 4 yes ZmSMR1 Functional validation of spider mite resistance, where B represents the correlation between genotype and spider mite damage level.

[0031] Figure 5 This characterizes the physical and chemical properties of the leaves and cell walls of Jing92, J2416, and the KO series. Figure 6 yes ZmSMR1 Map of gene overexpression vectors; Figure 7 It shows ZmSMR1 The nucleotide sequence of a gene; Figure 8 The results show the phenotypes of "Jing 92" and "Jing 2416" after inoculation, as well as the number of spider mites per plant.

[0032] Figure 9 This is a graph showing the phenotypic analysis of spider mite damage levels and leaf thickness in 318 recurrent populations generated from the hybridization of "Jing 92" and "Jing 2416". Figure 10 Candidate genes for resistance to spider mites in the Jing92 strain ( ZmSMR2 Cloning, expression levels, and subcellular localization analysis of ) Figure 11 yes ZmSMR2 Figures related to functional validation of spider mite resistance; Figure 12 yes ZmSMR2 Map of gene overexpression vectors; Figure 13 It shows ZmSMR2 The nucleotide sequence of a gene; Figure 14 The phenotypes of "Jing 92" and "Jing 2416" under spider mite infestation and the number of spider mites per plant; Figure 15This is a graph showing the phenotypic analysis of spider mite damage levels and leaf thickness in 318 recurrent populations generated from the hybridization of "Jing 92" and "Jing 2416". Figure 16 Candidate genes for resistance to spider mites in the Jing92 strain ( ZmSMR3 Cloning, expression levels, and subcellular localization analysis of ) Figure 17 yes ZmSMR3 Figures related to functional validation of spider mite resistance; Figure 18 yes ZmSMR3 Map of gene overexpression vectors; Figure 19 It shows ZmSMR3 The nucleotide sequence of a gene. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0034] ZmSMR1 Application in maize germplasm creation and variety breeding Materials and Methods Jing92 and Jing2416 are both improved maize inbred lines of the Huangzao IV variety bred by the Maize Research Institute of Beijing Academy of Agricultural and Forestry Sciences.

[0035] QTL localization: Construct a population of recombinant inbred lines (RILs) and localize QTL qSMR3-1.

[0036] CRISPR-Cas9 was used to knock out 92 cells. ZmSMR1 We constructed an overexpression vector to transform maize; and identified the transgenic plants by spider mite inoculation and determined their cell wall components.

[0037] Using Jing2416 × Jing92 as parents, a population of 318 recombinant inbred lines (RILs) was constructed (Wang et al., 2025). Resequencing of the RIL population yielded 2,154 genetic bins, from which a genetic linkage map was constructed. The RILs were planted twice, in Hainan and Beijing, and named 21HN and 22TZ, respectively. A randomized complete block design was used, with 18 plants per line planted per row, 5 m long, 60 cm between rows, and 27.5 cm between plants. The planting area was covered with 40-mesh insect-proof netting to prevent spider mite escape and reduce the impact of rainfall. Spider mites were reared in the laboratory using common bean; at the maize tasseling stage, a bean leaf infested with mites was inoculated onto the base of the third maize leaf from the bottom. Fifteen days after inoculation, the spider mite infestation level on each individual plant was assessed according to the previously described method (Di et al., 2024). Correlation analysis and distribution frequency analysis using nonlinear Gaussian regression in GraphPad Prism 5 (http: / / www.graphpad.com / ) were employed to assess the correlation and distribution of spider mite damage levels under two environmental conditions. Windows QTL Cartographer 2.5 software was used for QTL localization with composite interval mapping (CIM) at 1 cm steps and standard model controls. The LOD threshold was determined at a significance level of 0.05 using a permutation test after 1,000 iterations.

[0038] Precise positioning An F2 population of 1,500 individuals was constructed using two RILs that exhibited high resistance (007) and high susceptibility (009) to spider mites for fine mapping. The major-effect QTLs were then used. qSMR3-1 Internally, 25 SNPs were identified between the two parents, with approximately equal spacing between adjacent markers. This population was planted in Beijing on May 15, 2023, and leaf samples were collected for DNA extraction. Genotyping of the population was performed using targeted sequencing (Target-Seq) following the method of Campbell et al. (2015) on the Illumina HiSeq platform, with paired-end 150 bp sequences. The primers used are listed in Table S1. Spider mite resistance was assessed as above. Fine mapping was then performed using the CIM mode in WinQTLcart software, and the LOD threshold was determined through a 1,000-permutation test as described above.

[0039] The primers used in each operation are listed in Table S1. Competitive allele-specific PCR (KASP) marker detection KASP assay was designed based on functional SNPs within the Zm00001d042333 gene. Following the method of Shi et al. (2021), KASP assays were performed using DNA extracted from 1,500 individual plants from the 009 × 007 F2 population. A general linear model in SPSS software was used to assess the correlation between SNP genotypes and spider mite damage levels.

[0040] Candidate gene knockout and functional analysis Five target sites were designed for candidate genes using the Integrated CRISPR Target Design Suite (ICTDS) software developed by the Maize Research Institute of the Beijing Academy of Agricultural and Forestry Sciences. This software integrates target design, sgRNA secondary structure prediction, potential off-target identification, high off-target risk target removal, and characterization of positive plant mutations. The target sequences were cloned into the BsaI site of the PY5083 vector backbone using rCutSmart buffer (New England Biolabs, MA, USA). The resulting vector was used for transient transformation of maize protoplasts to verify editing efficiency. The two target sites with the highest editing efficiency, TS1 and ATS2, were selected and cloned between the AscI and ASISI sites of the PY5981 vector backbone to construct binary CRISPR / Cas9 vectors, named PG0038 and PG0039, respectively.

[0041] Genetic transformation of the maize inbred line Jing92 was performed according to the method of Zhang et al. (2021). The simplified steps are as follows: Two validated vectors were transformed into Agrobacterium tumefaciens strain EAH105, and positive clones were identified using phosphog-mannose isomerase (PMI) specific primers. Callus tissue of Jing92 was transformed using the Agrobacterium-mediated transformation method of Ishida et al. (2007), and regenerated tissues were screened on PMI-supplemented medium. Genomic DNA was extracted from the regenerated seedlings, and plants amplifying a 1.2 kb fragment using PMI specific primers were identified as positive T0 transgenic plants. DNA was extracted from T0 plants, the target fragment was amplified, and next-generation sequencing was performed. ICTDS was used to analyze mutation patterns. All positive T0 plants were self-pollinated to obtain T1 seeds. All primers used for vector construction and sequencing validation are shown in Table S1. To identify spider mite resistance, homozygous knockout plants were cultured in an insect-free greenhouse to stage V18, inoculated with spider mites, and the spider mite damage level was recorded according to the previous method (Di et al., 2024).

[0042] Candidate gene overexpression and functional analysis For candidate genes, the pCAMBIA3300 vector was modified to clone the CDS sequence of the target gene into the BamHI and SacI sites of the pCAMBIA3300 vector backbone. The resulting vector was used for transient transformation of maize protoplasts to verify transformation efficiency. ZmSMR1 Overexpression vector map such as Figure 6 As shown.

[0043] The vector plasmid was transferred into Agrobacterium EHA105 via electroporation, and identified by PCR. Using freshly peeled corn embryos (approximately 1 mm in diameter), each embryo was placed in a 2 ml plastic centrifuge tube containing 1.8 mL of suspension. Approximately 150 immature embryos were processed within 30 minutes. The suspension was then removed, leaving the corn embryos in the tube. 1.0 ml of Agrobacterium suspension was added to the tube, and the tube was incubated for 5 minutes. The embryos in the centrifuge tube were then resuspended and transferred to a co-culture medium. Excess Agrobacterium suspension was removed from the surface using a pipette, and the tubes were co-cultured in the dark at 23°C for 3 days. After co-culture, the embryos were transferred to resting medium and incubated in the dark at 28°C for 6 days. They were then transferred to selection medium containing diammonium phosphate for two weeks of selection culture, followed by two weeks of selection culture on a fresh selection medium. Resistant callus tissue was transferred to differentiation medium and cultured at 25°C, 5000 lx, under light for 3 weeks. The differentiated seedlings were then transferred to rooting medium and cultured at 25°C, 5000 lx, under light until rooting. The seedlings were then transferred to small pots for further growth, and after a certain growth stage, transplanted into a greenhouse. Seeds were harvested 3-4 months later. To identify spider mite resistance, overexpressing plants were cultured to stage V18 in an insect-free greenhouse, inoculated with spider mites, and the spider mite damage level was recorded according to a previous method (Di et al., 2024).

[0044] Blade thickness analysis At the tasseling stage, spike-side leaves were collected from the RIL population and transgenic plants. Small pieces (1 × 0.5 cm) were cut from the middle of the leaves and preserved in FAA fixative (Servicebio, Shanghai, China). Samples were dehydrated with graded ethanol and then embedded in paraffin. The embedded blocks were sliced ​​into 5 μm thick sections using a microtome, and the slides were dried in a 60℃ oven. After dewaxing, the sections were stained with safranin-fast green for approximately 2 minutes, washed with water, and dried at 60℃. The sections were cleared in fresh xylene for 5 minutes and mounted with neutral resin. Images were acquired under an optical microscope using CaseViewer software, and leaf thickness was measured at 30x magnification. The correlation analysis function in GraphPad Prism 5 was used to analyze the correlation between average leaf thickness and spider mite infestation level, and the two-tailed p-value was calculated.

[0045] ZmSMR1 Subcellular localization Using Jing92 cDNA as a template, PCR amplification was performed. ZmSMR1 The full-length coding sequence of the fusion protein was obtained and then cloned into the pCAMBIAsuper1300-GFP expression vector. To observe the localization of the fusion protein in living cells, the recombinant plasmid was introduced into maize mesophyll protoplasts using a polyethylene glycol-mediated transfection method. For precise localization, co-transfection with Golgi apparatus and endoplasmic reticulum markers tagged with mCherry was performed. After transfection, protoplasts were cultured at 28°C for 16–24 hours to achieve stable expression. Protoplasts exhibiting stable fluorescence were observed under a confocal laser scanning microscope (Stellaris 5, Leica Microsystems). GFP and mCherry signals were acquired using a standard filter set, followed by image channel merging and signal analysis (Fu et al., 2023).

[0046] Transmission electron microscopy observation The tissue block (approximately 1 mm) 3 Immediately fix with 2.5% glutaraldehyde for 24 hours. After fixation, wash with phosphate-buffered saline (PBS) for 6 hours, then fix with 1% osmium tetroxide for 2 hours. Subsequently, dehydrate using a gradient of ethanol: 30% ethanol for 10 minutes, 50% ethanol for 10 minutes, 70% ethanol (containing uranium acetate) for 3 hours, 80% ethanol for 10 minutes, 95% ethanol for 15 minutes, and twice with 100% ethanol (50 minutes each time), followed immediately by immersion in propylene oxide for 30 minutes. Place the sample in a 1:1 mixture of propylene oxide and epoxy resin for 2 hours, then in pure epoxy resin for 3 hours, and then embed in fresh pure epoxy resin and polymerize at 72°C for 24 hours. Cut the embedded blocks into 70 nm thick ultrathin sections using an ultramicrotome (UC-7, Leica). After lead staining, the sections were observed using a transmission electron microscope (JEM1400, Japan).

[0047] Cell wall monosaccharide content determination Following previous methods (Jayamanohar et al., 2019; Wang et al., 2026), the monosaccharide composition was analyzed using high-performance liquid chromatography (HPLC). The simplified procedure is as follows: the sample was hydrolyzed with 72% sulfuric acid at 30°C for 1 hour, diluted to 10 mL, and then hydrolyzed again at 110°C for 2 hours under nitrogen. After neutralization, the hydrolysis product was derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) at 70°C for 60 minutes. The derivative was extracted with chloroform, and the aqueous phase was filtered through a 0.45 μm filter before analysis. An Agilent 1200 series HPLC system equipped with a C18 column (4.6 mm × 250 mm, 5 μm) was used. The mobile phase consisted of 15% acetonitrile (A) and 40% acetonitrile (B) (both diluted with 0.05 mol / L KH₂PO₄, pH 6.8), and the flow rate was 1.0 mL / min. The gradient elution program was set as follows: 0-10 minutes, 100% A; 10-40 minutes, 92% A; 40-45 minutes, 63% A; 45-50 minutes, 100% A. The column temperature was maintained at 25°C, and the detection wavelength was 254 nm.

[0048] Example 1. High mite resistance of Jing 92 and its response to spider mite infestation. Two improved Huangzao IV lines—Jing92 and Jing2416—showed different leaf characteristics 10 days after spider mite infestation. Jing92's leaves remained relatively green with limited damage, while Jing2416's leaves exhibited yellowing and wilting, accompanied by severe necrotic spots. Figure 1 The phenotypic quantification results also showed that the leaf damage rating and spider mite count of Jing 92 were significantly lower than those of Jing 2416. Figure 1 BC in the figure represents the damage level and number of spider mites in “Jing 92” and “Jing 2416” cultivated in a greenhouse 7 days after inoculation. The data are expressed as the average value ± standard error of 30 individual plants.

[0049] Example 2. Correlation between maize spider mite resistance and leaf thickness Spider mite infestation was observed in all RIL lines, but the severity varied (e.g., Figure 2 Image A in the figure represents representative images of resistant and susceptible plants in the population 15 days after spider mite infection. In resistant lines, the damaged area on the third leaf remains green, with lesion coverage less than 30%; while in susceptible lines, the leaves show withered edges, and lesion coverage exceeds 70%. Maize Jing 92 exhibited relatively strong spider mite resistance in both planting seasons. Figure 2In the figure, B represents the spider mite damage level of the parental and recurrent populations under two different planting conditions. The histogram represents the mean ± standard error of 20 parental lines and 318 recurrent populations (each population has three to five replicates). Maize Jing 2416 is more susceptible to the disease. The average resistance rating of the RIL population is between that of the parents, indicating that there is an additive effect on spider mite resistance.

[0050] Based on the fact that the leaf thickness of Jing 92 was significantly greater than that of the susceptible inbred line (Diet al., 2024), the thickness of the ear-position leaf was measured to assess whether this physiological trait plays a role in spider mite resistance throughout the RIL population. The measured leaf thickness varied considerably, ranging from 141.7 to 324 µm. Figure 2 C, where C is a representative image of the leaf cross-section of the recurrent population under an optical microscope at 30x magnification (upper and lower: representative cross-sections of relatively thick and thin leaves), and shows a significant negative correlation with the spider mite infestation level. The two-tailed p-value of the Pearson correlation coefficient is less than 0.0001, indicating that it is significant. r The value is -0.63 ( Figure 2 The correlation and linear regression analysis of average leaf thickness and spider mite damage level in the recurrent population (D, where D is the correlation between average leaf thickness and spider mite damage level) indicates that the thicker the leaf, the stronger the resistance to spider mites.

[0051] Example 3. Major QTL localization and candidate gene cloning Using the genetic map constructed for the Jing 2416 × Jing 92RIL population (Wang et al., 2024), a total of 6 QTLs associated with spider mite resistance were identified over two growing seasons (Table 1 and 2024). Figure 3 In this context, A represents the chromosomal location of the QTL for resistance to spider mites found throughout the genome, along with its corresponding log-likelihood ratio (LOD) value. Location was determined using a genetic map constructed for the RIL population, with the LOD cutoff value determined through 1000 permutations at a significance level of 0.05. Specifically, QTL- qSMR3- 1 It was located on chromosome 3 (Chr 3) and detected in both growing seasons, with phenotypic variation explained (%PVE) of 14.3% in Hainan and 5.3% in Beijing. qSMR3-1 As a major-effect QTL identified in Hainan, it covers a 15Mb genomic region and contains 237 genes.

[0052] Table 1. QTLs regulating spider mite resistance were identified using RILs constructed from Jing92 × Jing 2416 hybridization. aQTLs are named according to the general rules (McCouch et al. 1997), starting with the lowercase letter q, followed by the trait name and the chromosome number where the locus is located; when multiple loci are located on the same chromosome, they are distinguished by -1, -2, etc.

[0053] b % PVE: Phenotypic contribution rate explained by a single quantitative trait locus.

[0054] c Additive effect: A positive value indicates that the Jing92 allele has an synergistic effect, while a negative value indicates that the Jing2416 allele can enhance the effect of the target trait.

[0055] To further refine the positioning qSMR3-1 QTL intervals were identified and candidate genes controlling spider mite resistance were determined. An F2 population was constructed by hybridizing two phenotypic extreme recombinant inbred lines (RILs) – 007 (highly resistant) and 009 (highly susceptible). These two inbred lines were used only in… qSMR3-1 The haplotypes of the two QTLs differed, while they shared the same haplotype across all other minor-effect QTLs. Sixteen SNP markers were developed using the polymorphism of Jing92 and Jing2416 within a 15 Mb target region. Genotypic and phenotypic analyses of 2,000 F2 single plants ultimately narrowed the QTLs down to a 1 Mb interval between markers ID17 and ID19. Figure 3 In the diagram, B represents the SNP markers 3 and 16 between two parents within the 15M region of the major QTL qSMR3-1, finely mapped using an F2 population of 2000 individuals from highly resistant and susceptible RILs. Red bars indicate the 1M region containing spider mite resistance loci. In the Jing92 genome, this region contains 10 annotated genes (Wang et al., 2023). ZmSMR1 The background and induced transcription levels of the gene in Jing92 were significantly higher than those in Jing2416. Figure 3 The C in the text refers to the concentration of C in the leaves of the two parent plants before and after spider mite infection. ZmSMR1 The relative expression is represented by the mean ± standard error for each biological replicate (5 individual plants per replicate). and (p<0.05 and p<0.001, respectively). Furthermore, this gene was upregulated 7-fold in Jing92 after spider mite infection, but only 2-fold in Jing2416. Sequence analysis of this gene showed that, compared to Jing2416, Jing92 had a 364bp deletion 54bp upstream of the ATG region, and a non-synonymous SNP from G to A in the coding region (…). Figure 3D in it, where D is the 364-base pair deletion in the promoter region of the Jing92 gene and the non-synonymous single nucleotide polymorphisms in its coding sequence). Therefore, this gene was identified as a candidate gene conferring resistance to spider mites and named SPIDER MITE RESISTANCE 1 (SMR1) . ZmSMR1 was annotated as a member of glycosyltransferase family 47 (GT47) (Zhang et al., 2023; Zhong & Ye, 2003), indicating that it may be involved in the biosynthesis of cell wall polysaccharides. To evaluate its biological function, ZmSMR1 -eGFP fusion protein was transiently expressed in maize protoplasts to determine its subcellular localization. The results showed that ZmSMR1 was localized to the endoplasmic reticulum and Golgi apparatus ( Figure 3 E in it, where E is the subcellular localization of the transient overexpressed ZmSMR1 -GFP fusion protein in maize leaf protoplasts. The markers of the endoplasmic reticulum and Golgi apparatus combined with mCherry were co-transformed, and GFP and mCherry fluorescence were shown in green and purple respectively. The images were taken about 16 hours after transfection, and the bar represents 5 micrometers), which is consistent with its expected function as a glycosyltransferase involved in the biosynthesis of cell wall polysaccharides.

[0056] Example 4. ZmSMR1 Functional verification in terms of resistance to spider mites For ZmSMR1 , a KASP marker was developed for the non-synonymous SNP located in exon 2, and the F2 population of 009 (susceptible RIL) × 007 (resistant RIL) was used for genotyping. The detection results showed three distinct clusters ( Figure 4 A in it, where A is ZmSMR1 the KASP detection of the non-synonymous single nucleotide polymorphism [G / A] in exon 2. The genotypes of each cluster are shown in the figure, and the pink dots represent the no-template control and ambiguous data points respectively). Jing92 is the GG allele, and Jing2416 is the AA allele. The F2 individuals carrying the GG homozygote were significantly more resistant to spider mites than those carrying the AG and AA alleles ( Figure 4 B in it, where B is the correlation between the genotype and the spider mite damage level; the data represent the mean ± standard error of all F2 individuals with the same genotype; C-F are the development and characterization of the CRISPR-Cas9 knockout mutants of ZmSMR1 in Jing92). There were significant differences in the spider mite damage levels between the heterozygote (AG) and the GG homozygote and the AA homozygote, indicating that the GG allele from Jing92 may be a semi-dominant locus and is involved in spider mite resistance in a dose-dependent manner.

[0057] To further study ZmSMR1To investigate its anti-mite function, a CRISPR-Cas9 knockout (KO) line was created in the Jing92 context. Five target sites were designed and transiently transformed into Jing92 protoplasts to evaluate editing efficiency. The two target sites with the highest editing efficiency, TS1 and ATS2, were selected and cloned into binary vectors (PG0038 and PG0039). Figure 3a As shown in A and B in the diagram; Figure 4 C in the text is ZmSMR1 A schematic diagram of the gene structure, CRISPR target sites, and the two resulting knockout (KO) alleles (with deletions of 10 and 13 base pairs, respectively; TS1 and ATS2 indicate two target sites on exon 2), was introduced into Jing92. Two independent mutant alleles were obtained, with deletions of 10 bp and 13 bp in exon 2, respectively, resulting in frameshift and protein truncation, and were named... ZmSMR1 9r1 and ZmSMR1 9r2 Compared to the Beijing 92, ZmSMR1 9r1 and ZmSMR1 9r2 Homozygotes showed a significantly increased susceptibility to spider mite infestation, exhibiting more severe leaf damage and chlorophyll loss. Figure 4 D in the figure represents representative leaves of Jing92 and the two KO lines 7 days after spider mite infection in the seedling stage (scale bar 2 cm). Consistent with this phenotype, the damage rating of both knockout lines was significantly increased. Figure 4 In the figure, E represents the spider mite infestation level of the KO line cultured in the greenhouse during the seedling stage, and the number of spider mites per plant also increased significantly. Figure 4 In the figure, F represents the number of spider mites on each wild-type and KO lineage plant, confirming... ZmSMR1 It is a key contributing factor to the resistance of spider mites under the genetic background of Jing 92.

[0058] Overexpression in mite-infected strains ZmSMR1 Subsequently, the overexpressing plants exhibited stronger resistance to spider mites, with reduced leaf damage and chlorophyll loss. Consistent with this phenotype, the spider mite infestation level of the overexpressing lines was significantly increased, and the number of spider mites per plant was extremely significantly reduced. This confirms... ZmSMR1 Key contributing factors to spider mite resistance, such as Figure 4 In the figures, D, G, and H represent the spider mite infestation level of the KO line cultured in the greenhouse during the seedling stage; H represents the number of spider mites on each plant, both wild-type and KO line. The data represent the mean ± standard deviation of 30 individuals. p<0.05; p < 0.001.

[0059] Example 5. ZmSMR1 Tetranychus resistance in knockout transgenic lines Homozygous knockout plants were cultured in an insect-free greenhouse until stage V18, then inoculated with spider mites, and the level of spider mite damage was recorded.

[0060] Compared with the wild-type Jing 92, homozygous knockout plants ZmSMR1 cr The thickness of the leaves at the ear position of the plant was significantly reduced. Figure 5 In the figures A and B, A is a representative leaf cross-sectional image of the Jing92, Jing2416, and KO series at 30x magnification (scale bar: 50 micrometers); B is the leaf thickness of field-grown plants at the V18 stage (histogram representing the mean ± standard deviation of 30 measurements from 5 biological replicates), but it is comparable to Jing2416, which further validates the... ZmSMR1 There is a potential mechanistic link between mediated leaf thickness and spider mite resistance. Given the role of the GT47 family in cell wall structure, cell wall thickness and cell packing density were measured in the mutant system. The results showed that, compared with the wild-type Jing92, the cell wall thickness of the mutant system was significantly reduced (…). Figure 5 In the figures C and D, C is a representative transmission electron microscope image of leaves harvested at the V18 stage, with red asterisks indicating cell walls (scale bar 1 micrometer); D is the cell wall thickness analysis (data expressed as mean ± standard deviation of 40 measurements). Since pectin and hemicellulose are key factors affecting cell wall thickness and cell packing density, quantitative analysis of cell wall components was performed to investigate... ZmSMR1 Effects on cell wall remodeling. Results showed that, compared to Jing92, the cell wall pectin and hemicellulose levels in both mutant systems were significantly reduced, falling to the level of Jing2416 (…). Figure 5 The E content (where E represents the lignin, pectin, and hemicellulose content in the cell walls of the KO series) showed no significant difference in lignin levels. Analysis of cell wall monosaccharide content indicated that... ZmSMR1 The mutation resulted in a significant decrease in the content of rhamnose and galacturonic acid, components of pectin and hemicellulose, by approximately one-third and one-quarter, respectively, while the levels of other monosaccharides showed no significant difference. Figure 5 In this context, F represents the monosaccharide content level in the cell walls of different genotypes, and the data represent the mean ± standard deviation of four biological replicates. (p<0.001). In summary, compared with Jing 92, the content of pectin and hemicellulose in the knockout lines was significantly reduced, as were the contents of rhamnose and galacturonic acid, indicating that... ZmSMR1 It is possible that changes in leaf physical properties are mediated through cell wall remodeling, thereby regulating spider mite resistance.

[0061] Example 6 For candidate genes, the pCAMBIA3300 vector was modified to clone the CDS sequence of the target gene into the BamHI and SacI sites of the pCAMBIA3300 vector backbone. The resulting vector was used for transient transformation of maize protoplasts to verify the transformation efficiency.

[0062] The vector plasmid was transferred into Agrobacterium EHA105 via electroporation, and identified by PCR. Using freshly peeled corn embryos (approximately 1 mm in diameter), each embryo was placed in a 2 ml plastic centrifuge tube containing 1.8 mL of suspension. Approximately 150 immature embryos were processed within 30 minutes. The suspension was then removed, leaving the corn embryos in the tube. 1.0 ml of Agrobacterium suspension was added to the tube, and the tube was incubated for 5 minutes. The embryos in the centrifuge tube were then resuspended and transferred to a co-culture medium. Excess Agrobacterium suspension was removed from the surface using a pipette, and the tubes were co-cultured in the dark at 23°C for 3 days. After co-culture, the embryos were transferred to resting medium and incubated in the dark at 28°C for 6 days. They were then transferred to selection medium containing diammonium phosphate for two weeks of selection culture, followed by two weeks of selection culture on a fresh selection medium. Resistant callus tissue was transferred to differentiation medium and cultured at 25°C, 5000 lx, under light for 3 weeks. The differentiated seedlings were then transferred to rooting medium and cultured at 25°C, 5000 lx, under light until rooting occurred. The seedlings were then transferred to small pots for further growth, and after a certain growth stage, transplanted into a greenhouse. Seeds were harvested 3-4 months later. To identify spider mite resistance, overexpressing plants were cultured to stage V18 in an insect-free greenhouse, inoculated with spider mites, and the spider mite damage level was recorded according to a previous method (Dietal., 2024).

[0063] ZmSMR1 The nucleotide sequence (SEQ ID NO.1) of the gene (gene number Zm00001d042333) is as follows (blue background indicates the CDS region, such as...). Figure 7 ZmSMR1 The amino acid sequence (SEQ ID NO.2) of the gene (gene number Zm00001d042333) is as follows: MGAPSSRALGAAFLLLLVALPSAFLYLTSSAATRATLLDLKPFSARCAPAVAPLRVFMYDLPARFHVAMMGAAAGAGFPAWPPSAGGIRRQHSVEYWMMASLQDGGAGPERGREAVRVRDPDAADAFFVPFFSSLSFNVHGRNMTDPDTEADRLLQVELVDILWKSKYWQRSAGRDHVIPMHHPNAFRFLRAMVNASILIVSDFGRYTKEL ASLRKDVVAPYVHVVGSFLDDDPPDPFEARHTLLFFRGRTVRKDEGKIRSKLEKILKGKEGVRFEDSIATGDGINISTEGMRSSKFCLHPAGDTPSSCRLFDAIVSH CVPVIVSSRIELPFEDEIDYSEFSLFFSVEEALRPDYLLNELRQVPKRKWVDMWLKLKNVSHHYEFQYPPRKGDAVNMIWRQVRHKIPAVNLAIHRNRRLKIPDWWG ZmSMR2 Application in maize germplasm creation and variety breeding Materials and Methods Jing92 and Jing2416 are both improved maize inbred lines of the Huangzao IV variety bred by the Maize Research Institute of Beijing Academy of Agricultural and Forestry Sciences.

[0064] Jing92 and Jing2416 are both improved maize inbred lines of the Huangzao IV variety bred by the Maize Research Institute of Beijing Academy of Agricultural and Forestry Sciences.

[0065] QTL localization: Construct a population of recombinant inbred lines (RILs) and localize QTL qSMR3-1.

[0066] CRISPR-Cas9 was used to knock out 92 cells. ZmSMR2 We constructed an overexpression vector to transform maize; and identified the transgenic plants by spider mite inoculation and determined their cell wall components.

[0067] Using Jing2416 × Jing92 as parents, a population of 318 recombinant inbred lines (RILs) was constructed (Wang et al., 2025). Resequencing of the RIL population yielded 2,154 genetic bins, from which a genetic linkage map was constructed. The RILs were planted twice, in Hainan and Beijing, and named 21HN and 22TZ, respectively. A randomized complete block design was used, with 18 plants per line planted per row, 5 m long, 60 cm between rows, and 27.5 cm between plants. The planting area was covered with 40-mesh insect-proof netting to prevent spider mite escape and reduce the impact of rainfall. Spider mites were reared in the laboratory using common bean; at the maize tasseling stage, a bean leaf infested with mites was inoculated at the base of the third maize leaf from the bottom. Fifteen days after inoculation, the spider mite infestation level on each individual plant was assessed according to the previously described method (Di et al., 2024). Correlation analysis and distribution frequency analysis using nonlinear Gaussian regression in GraphPad Prism 5 (http: / / www.graphpad.com / ) were employed to assess the correlation and distribution of spider mite damage levels under two environmental conditions. Windows QTL Cartographer 2.5 software was used for QTL localization with composite interval mapping (CIM) at 1 cm steps and standard model controls. The LOD threshold was determined at a significance level of 0.05 using a permutation test after 1,000 iterations.

[0068] Precise positioning An F2 population of 1,500 individuals was constructed using two RILs that exhibited high resistance (007) and high susceptibility (009) to spider mites for fine mapping. The major-effect QTLs were then used. qSMR3-1 Internally, 25 SNPs were identified between the two parents, with approximately equal spacing between adjacent markers. This population was planted in Beijing on May 15, 2023, and leaf samples were collected for DNA extraction. Genotyping of the population was performed using targeted sequencing (Target-Seq) on the Illumina HiSeq platform, with paired-end 150 bp, following the method of Campbell et al. (2015). Spider mite resistance was assessed as described above. Fine mapping was then performed using the CIM mode of WinQTLcart software, and the LOD threshold was determined through a 1,000-permutation test as described above.

[0069] Competitive allele-specific PCR (KASP) marker detection KASP detection was designed based on functional SNPs within the Zm00001eb143220 gene. Following the method of Shi et al. (2021), KASP detection was performed using DNA extracted from 1,500 individual plants from the 009 × 007 F2 population. KASP detection primer sequences are shown in Table S2-1. A general linear model in SPSS software was used to assess the correlation between SNP genotypes and spider mite damage levels.

[0070] Table S2-1. Primers used in each operation Candidate gene knockout and functional analysis Five target sites were designed for candidate genes using the Integrated CRISPR Target Design Suite (ICTDS) software developed by the Maize Research Institute of the Beijing Academy of Agricultural and Forestry Sciences. This software integrates target design, sgRNA secondary structure prediction, potential off-target identification, high off-target risk target removal, and characterization of positive plant mutations. The target sequences were cloned into the BsaI site of the PY5083 vector backbone using rCutSmart buffer (New England Biolabs, MA, USA). The resulting vector was used for transient transformation of maize protoplasts to verify editing efficiency. The two target sites with the highest editing efficiency, TS1 and ATS2, were selected and cloned between the AscI and ASISI sites of the PY5981 vector backbone to construct binary CRISPR / Cas9 vectors, named PG0038 and PG0039, respectively.

[0071] Genetic transformation of the maize inbred line Jing92 was performed according to the method of Zhang et al. (2021). The simplified steps are as follows: Two validated vectors were transformed into Agrobacterium tumefaciens strain EAH105, and positive clones were identified using phosphog-mannose isomerase (PMI) specific primers. Callus tissue of Jing92 was transformed using the Agrobacterium-mediated transformation method of Ishida et al. (2007), and regenerated tissues were screened on PMI-supplemented medium. Genomic DNA was extracted from the regenerated seedlings, and plants amplifying a 1.2 kb fragment using PMI specific primers were identified as positive T0 transgenic plants. DNA was extracted from T0 plants, the target fragment was amplified, and next-generation sequencing was performed. ICTDS was used to analyze mutation patterns. All positive T0 plants were self-pollinated to obtain T1 seeds. All primers used for vector construction and sequencing validation are shown in Table S2-1. To identify spider mite resistance, homozygous knockout plants were cultured in an insect-free greenhouse to stage V18, inoculated with spider mites, and the spider mite damage level was recorded according to the previous method (Di et al., 2024).

[0072] ZmSMR2 Subcellular localization Using Jing92 cDNA as a template, PCR amplification was performed. ZmSMR2 The full-length coding sequence of the fusion protein was obtained and then cloned into the pCAMBIAsuper1300-GFP expression vector. To observe the localization of the fusion protein in living cells, the recombinant plasmid was introduced into maize mesophyll protoplasts using a polyethylene glycol-mediated transfection method. For precise localization, co-transfection with Golgi apparatus and endoplasmic reticulum markers tagged with mCherry was performed. After transfection, protoplasts were cultured at 28 °C for 16–24 hours to achieve stable expression. Protoplasts exhibiting stable fluorescence were observed under a confocal laser scanning microscope (Stellaris 5, Leica Microsystems). GFP and mCherry signals were acquired using a standard filter set, followed by image channel merging and signal analysis (Fu et al., 2023).

[0073] Candidate gene overexpression and functional analysis For candidate genes, the pCAMBIA3300 vector was modified to clone the CDS sequence of the target gene into the pCAMBIA3300 vector backbone. The resulting vector was used for transient transformation of maize protoplasts to verify transformation efficiency. ZmSMR2 Overexpression vector map such as Figure 12 As shown.

[0074] The vector plasmid was transferred into Agrobacterium EHA105 via electroporation, and identified by PCR. Using freshly peeled corn embryos (approximately 1 mm in diameter), each embryo was placed in a 2 ml plastic centrifuge tube containing 1.8 mL of suspension. Approximately 150 immature embryos were processed within 30 minutes. The suspension was then removed, leaving the corn embryos in the tube. 1.0 ml of Agrobacterium suspension was added to the tube, and the tube was incubated for 5 minutes. The embryos in the centrifuge tube were then resuspended and transferred to a co-culture medium. Excess Agrobacterium suspension was removed from the surface using a pipette, and the tubes were co-cultured in the dark at 23°C for 3 days. After co-culture, the embryos were transferred to resting medium and incubated in the dark at 28°C for 6 days. They were then transferred to selection medium containing diammonium phosphate for two weeks of selection culture, followed by two weeks of selection culture on a fresh selection medium. Resistant callus tissue was transferred to differentiation medium and cultured at 25 °C, 5000 lx, under light for 3 weeks. The differentiated seedlings were then transferred to rooting medium and cultured at 25 °C, 5000 lx, under light until rooting occurred. The seedlings were then transferred to small pots for further growth, and after a certain growth stage, transplanted into a greenhouse. Seeds were harvested 3-4 months later. To identify spider mite resistance, overexpressing plants were cultured in an insect-free greenhouse to stage V18, inoculated with spider mites, and the spider mite damage level was recorded according to a previous method (Di et al., 2024).

[0075] Example 2-1. High mite resistance of Jing92 leaves and its response to spider mite infestation. Two improved strains of Huangzao IV, Jing92 and Jing2416, exhibited drastically different resistance to spider mites. Ten days after spider mite infestation, the leaves of Jing92 remained relatively green with limited damage; while the leaves of Jing2416 showed yellowing and wilting, accompanied by severe necrotic spots. Figure 8 In the image, A represents a representative image of leaves infected with spider mites 7 days after inoculation for both strains. The scale bar represents 2 cm. Phenotypic quantification results also showed that the leaf damage rating and spider mite count of Jing 92 were significantly lower than those of Jing 2416. Figure 8 BC in the figure represents the damage level and number of spider mites in “Jing 92” and “Jing 2416” cultivated in a greenhouse 7 days after inoculation. The data are expressed as the average value ± standard error of 30 individual plants.

[0076] Example 2-2. Correlation between maize spider mite resistance and leaf thickness Spider mite infestation was observed in all RIL lines, but the severity varied (e.g., Figure 9 In Figure A, A represents a representative image of resistant and susceptible plants from 318 recurrent populations generated from the cross between "Jing 92" and "Jing 2416" 15 days after spider mite infection. Overall, in resistant lines, the damaged area on the third leaf remained green, with lesion coverage less than 30%; while in susceptible lines, the leaves showed withered edges, and lesion coverage exceeded 70%. Jing 92 exhibited relatively strong spider mite resistance in both planting seasons (e.g., ...). Figure 9 In the figure, B represents the spider mite damage level of the two parents and the recurrent population under two different planting conditions. The histogram represents the mean ± standard error of 20 two-parent lines and 318 recurrent populations (each population has three to five replicates). The other parent, Jing 2416, is more susceptible to the disease. The average resistance rating of the RIL population is between that of the two parents, indicating that there is an additive effect on spider mite resistance.

[0077] Based on the fact that the leaf thickness of Jing 92 was significantly greater than that of the susceptible inbred line (Di et al., 2024), the thickness of the ear-position leaf was measured to assess whether this physiological trait plays a role in spider mite resistance throughout the RIL population. The measured leaf thickness varied considerably, ranging from 141.7 to 324 µm. Figure 9 C in the figure represents a representative image of a leaf cross-section of a recurrent population under an optical microscope at 30x magnification. (Upper and lower parts: representative cross-sections of relatively thick and thin leaves), and this image shows a significant negative correlation with the severity of spider mite infestation (Pearson). r The value is -0.63 ( Figure 9In the equation, D represents the correlation and linear regression analysis between the average leaf thickness in the recurrent population and the severity of spider mite damage. The two-tailed p-values ​​of the Pearson correlation coefficient are less than 0.0001, indicating that the correlation is significant, suggesting that thicker leaves are associated with stronger resistance to spider mites.

[0078] Examples 2-3. Major QTL localization and candidate gene cloning Using the genetic map constructed for the Jing 2416 × Jing 92 RIL population (Wang et al., 2024), a total of 6 QTLs associated with spider mite resistance were identified over two growing seasons, as shown in Table 2-1 and Figure 10 In this context, A (A represents the chromosomal location of a QTL for resistance to spider mites found throughout the genome and its corresponding log-likelihood ratio (LOD) value, located using a genetic map constructed for the RIL population, with the LOD cutoff value determined through 1000 permutations at a significance level of 0.05). A QTL- qSMR3-1 It was located on chromosome 3 (Chr 3) and detected in both growing seasons, with phenotypic explained rates (%PVE) of 14.3% and 5.3% in Hainan and Beijing, respectively. Other QTLs ( Figure 10 A) in the middle are named respectively qSMR3-2, qSMR4, qSMR5, qSMR6 and qSMR9 All were detected under only one growth condition, contributing 4.5%, 4.7%, 3.4%, 5.7%, and 3.2% to phenotypic variation, respectively (Table 2-1 and 2-2). Figure 10 The A in the figure indicates that these are minimal QTLs. qSMR3-1 As a major-effect QTL identified in Hainan, it covers a 15Mb genomic region and contains 237 genes.

[0079] Table 2-1. QTLs regulating spider mite resistance were identified using RILs constructed by crossing Jing92 × Jing2416. a QTLs are named according to the general rules (McCouch et al. 1997), starting with the lowercase letter q, followed by the trait name and the chromosome number where the locus is located; when multiple loci are located on the same chromosome, they are distinguished by -1, -2, etc.

[0080] b % PVE: Phenotypic contribution rate explained by a single quantitative trait locus.

[0081] c Additive effect: A positive value indicates that the Jing92 allele has an synergistic effect, while a negative value indicates that the Jing2416 allele can enhance the effect of the target trait.

[0082] To further refine the positioning qSMR3-1 To identify QTL sequences and candidate genes controlling spider mite resistance, we constructed an F2 population by crossing two phenotypic extreme recombinant inbred lines (RILs) – 007 (highly resistant) and 009 (highly susceptible). These two inbred lines were used only in… qSMR3-1 The haplotypes of the two QTLs differed, while they shared the same haplotype across all other minor-effect QTLs. Sixteen SNP markers were developed using the polymorphism of Jing92 and Jing2416 within a 15 Mb target region. Genotypic and phenotypic analyses of 2,000 F2 single plants ultimately narrowed the QTLs down to a 1 Mb interval between markers ID17 and ID19. Figure 10 In the diagram, B represents the SNP markers 3 and 16 between two parents within the 15M region of the major QTL qSMR3-1; fine mapping was performed using an F2 population of 2000 individuals from highly resistant and susceptible RILs. Red bars indicate the 1M region containing spider mite resistance loci. In the Jing92 genome, this region contains 10 annotated genes (Wang et al., 2023). ZmSMR2 The background and induced transcription levels of the gene in Jing92 were significantly higher than those in Jing2416. Figure 10 The C in the text refers to the concentration of C in the leaves of the two parent plants before and after spider mite infection. ZmSMR2 The relative expression; the data represent the mean ± standard error for each biological replicate (5 individual plants per replicate). and (These represent p < 0.05 and p < 0.001, respectively).

[0083] Furthermore, this gene was upregulated 5-fold in Jing92 after spider mite infection, while it was only upregulated 1.5-fold in Jing2416. Sequence analysis of this gene showed that, compared with Jing2416, Jing92 had two non-synonymous mutations in the coding region: A to G and T to G. (e.g.) Figure 10 In the D, D represents the ratio of the two parents. ZmSMR2 A schematic diagram of natural allelic variation. The 364-base pair deletion in the promoter region of the Jing92 gene and the non-synonymous single nucleotide polymorphisms in its coding sequence are shown below. Therefore, this gene was identified as a candidate gene conferring resistance to spider mites and named... SPIDER MITE RESISTANCE 2 (SMR2) . ZmSMR2Annotated as an exostosinlike gene, also known as a glycosyltransferase (Zhang et al., 2023; Zhong & Ye, 2003), it suggests a possible involvement in the biosynthesis of cell wall polysaccharides. To assess its biological function, it was transiently expressed in maize protoplasts. ZmSMR2 -eGFP fusion protein was used to determine its subcellular localization. The results showed that... ZmSMR2 Located in the endoplasmic reticulum and Golgi apparatus ( Figure 10 In the context of E, E represents transient overexpression. ZmSMR2 - Subcellular localization of the GFP fusion protein in maize leaf protoplasts; endoplasmic reticulum and Golgi apparatus markers bound to mCherry were co-transformed; GFP and mCherry fluorescence are shown in green and purple, respectively; images were taken approximately 16 hours after transfection (bars represent 5 micrometers), consistent with its expected function as a glycosyltransferase involved in the biosynthesis of cell wall polysaccharides.

[0084] Examples 2-4. ZmSMR2 Functional validation in terms of spider mite resistance To verify ZmSMR2 To investigate its anti-mite function, a CRISPR-Cas9 knockout (KO) line was created in the Jing92 context. Five target sites were designed and transiently transformed into Jing92 protoplasts to evaluate editing efficiency. The two target sites with the highest editing efficiency were selected, cloned into a binary vector, and introduced into Jing92. Two independent mutant alleles were obtained, leading to frameshift, premature termination, and protein truncation, named […]. ZmSMR2 cr Compared to the Beijing 92, ZmSMR2 cr Homozygous mutants showed significantly increased susceptibility to spider mite infestation, exhibiting more severe leaf damage and chlorophyll loss, OE- ZmSMR2 Overexpression plants are the opposite (e.g. Figure 11 In the A and C, A refers to Jing92 overexpression (OE-) 7 days after spider mite infection in the seedling stage. ZmSMR2 ) and knockout system ( ZmSMR2 cr (A representative leaf of the plant, scale bar 2 cm, where C is the number of spider mites on each plant), confirming... ZmSMR2 It is a key contributing factor to the resistance of spider mites under the genetic background of Jing 92.

[0085] In addition, overexpression in mite-susceptible strains ZmSMR2 Subsequently, the overexpressing plants exhibited stronger resistance to spider mites, with reduced leaf damage and chlorophyll loss. Consistent with this phenotype, the spider mite damage level of the overexpressing lines was significantly increased (e.g., Figure 11In the figure, B represents the spider mite infestation level for each plant, and the number of spider mites per plant is significantly reduced (e.g., ...). Figure 11 In the figure, C represents the number of spider mites per plant. ZmSMR2 It is a key contributing factor to spider mite resistance.

[0086] ZmSMR2 Nucleotide sequence (SEQ ID NO. 3) of gene (gene number Zm00001eb143220) (blue background indicates CDS region): ZmSMR2 The amino acid sequence (SEQ ID NO. 4) of the gene (gene number Zm00001eb143220) is as follows: MGLQKAYLRRIAFFVFEMWLATVFALLILFALASIGRSSRMLEKSYYTETEVASRGDSVTSQDPAESEFADLARLLPKVATDDRTVIITSVNEAFARPDSLLGLFRESFQAGEGIGHLLNNVLVVAVDAKAFSHCRAVHPHCYLLEVKTIDLSSANNYMSEAYIELVWTKLSLLQRILELGYNF LFTDVDIVWFRNPFRHISVFADMTTSSDVFYGDADGLDNWPNTGFFYVKATSRTVEMLRRWRAARFPANHEQAIFNDIKHELARDLGARVQFLDTARFGSFCRIFHIDMAAACTMHANCCVGLGNKLHDLRDVLRQWKNYTGLTPQAKKSQKFIWKDPAKCGTPDKRKRTHESEKCTTSASIV ZmSMR3 Application in maize germplasm creation and variety breeding Materials and Methods Jing92 and Jing2416 are both improved maize inbred lines of the Huangzao IV variety bred by the Maize Research Institute of Beijing Academy of Agricultural and Forestry Sciences.

[0087] Jing92 and Jing2416 are both improved maize inbred lines of the Huangzao IV variety bred by the Maize Research Institute of Beijing Academy of Agricultural and Forestry Sciences.

[0088] QTL localization: Construct a population of recombinant inbred lines (RILs) and localize QTL qSMR3-1.

[0089] CRISPR-Cas9 was used to knock out 92 cells. ZmSMR3 We constructed an overexpression vector to transform maize; and identified the transgenic plants by spider mite inoculation and determined their cell wall components.

[0090] Using Jing2416 × Jing92 as parents, a population of 318 recombinant inbred lines (RILs) was constructed (Wang et al., 2025). Resequencing of the RIL population yielded 2,154 genetic bins, from which a genetic linkage map was constructed. The RILs were planted twice, in Hainan and Beijing, and named 21HN and 22TZ, respectively. A randomized complete block design was used, with 18 plants per line planted per row, 5 m long, 60 cm between rows, and 27.5 cm between plants. The planting area was covered with 40-mesh insect-proof netting to prevent spider mite escape and reduce the impact of rainfall. Spider mites were reared in the laboratory using common bean; at the maize tasseling stage, a bean leaf infested with mites was inoculated at the base of the third maize leaf from the bottom. Fifteen days after inoculation, the spider mite infestation level on each individual plant was assessed according to the previously described method (Diet et al., 2024). Correlation analysis and distribution frequency analysis using nonlinear Gaussian regression in GraphPad Prism 5 (http: / / www.graphpad.com / ) were employed to assess the correlation and distribution of spider mite damage levels under two environmental conditions. Windows QTL Cartographer 2.5 software was used for QTL localization with composite interval mapping (CIM) at 1 cm steps and standard model controls. The LOD threshold was determined at a significance level of 0.05 using a permutation test after 1,000 iterations.

[0091] Precise positioning An F2 population of 1,500 individuals was constructed using two RILs that exhibited high resistance (007) and high susceptibility (009) to spider mites for fine mapping. The major-effect QTLs were then used. qSMR3-1 Internally, 25 SNPs were identified between the two parents, with approximately equal spacing between adjacent markers. This population was planted in Beijing on May 15, 2023, and leaf samples were collected for DNA extraction. Genotyping of the population was performed using targeted sequencing (Target-Seq) on the Illumina HiSeq platform, with paired-end 150 bp, following the method of Campbell et al. (2015). Spider mite resistance was assessed as described above. Fine mapping was then performed using the CIM mode of WinQTLcart software, and the LOD threshold was determined through a 1,000-permutation test as described above.

[0092] Competitive allele-specific PCR (KASP) marker detection KASP detection was designed based on functional SNPs within the Zm00001d042333 gene. Following the method of Shi et al. (2021), KASP detection was performed using DNA extracted from 1,500 individual plants from the 009 × 007 F2 population. KASP detection primer sequences are shown in Table S3-1. The correlation between SNP genotypes and spider mite damage severity was assessed using a general linear model in SPSS software.

[0093] Table S3-1. Primers used in each operation Competitive allele-specific PCR (KASP) marker detection KASP assay was designed based on functional SNPs within the Zm00001d042337 gene. Following the method of Shi et al. (2021), KASP assays were performed using DNA extracted from 1,500 individual plants from the 009 × 007 F2 population. A general linear model in SPSS software was used to assess the correlation between SNP genotypes and spider mite damage levels.

[0094] Candidate gene knockout and functional analysis Five target sites were designed for candidate genes using the Integrated CRISPR Target Design Suite (ICTDS) software developed by the Maize Research Institute of the Beijing Academy of Agricultural and Forestry Sciences. This software integrates target design, sgRNA secondary structure prediction, potential off-target identification, high off-target risk target removal, and characterization of positive plant mutations. The target sequences were cloned into the BsaI site of the PY5083 vector backbone using rCutSmart buffer (New England Biolabs, MA, USA). The resulting vector was used for transient transformation of maize protoplasts to verify editing efficiency. The two target sites with the highest editing efficiency, TS1 and ATS2, were selected and cloned between the AscI and ASISI sites of the PY5981 vector backbone to construct binary CRISPR / Cas9 vectors, named PG0038 and PG0039, respectively.

[0095] Genetic transformation of the maize inbred line Jing92 was performed according to the method of Zhang et al. (2021). The simplified steps are as follows: Two validated vectors were transformed into Agrobacterium tumefaciens strain EAH105, and positive clones were identified using phosphog-mannose isomerase (PMI) specific primers. Callus tissue of Jing92 was transformed using the Agrobacterium-mediated transformation method of Ishida et al. (2007), and regenerated tissues were screened on PMI-supplemented medium. Genomic DNA was extracted from the regenerated seedlings, and plants amplifying a 1.2 kb fragment using PMI-specific primers were identified as positive T0 transgenic plants. DNA was extracted from the T0 plants, the target fragment was amplified, and next-generation sequencing was performed. ICTDS was used to analyze mutation patterns. All positive T0 plants were self-pollinated to obtain T1 seeds. All primers used for vector construction and sequencing validation are shown in Table S3-1. To identify spider mite resistance, homozygous knockout plants were cultured in an insect-free greenhouse to stage V18, inoculated with spider mites, and the spider mite damage level was recorded according to the previous method (Di et al., 2024).

[0096] Blade thickness analysis At the tasseling stage, spike-position leaves were collected from the RIL population and transgenic plants. Small pieces (1 × 0.5 cm) were cut from the middle of the leaves and preserved in FAA fixative (Servicebio, Shanghai, China). Samples were dehydrated with graded ethanol and then embedded in paraffin. The embedded blocks were sliced ​​into 5 μm thick sections using a microtome, and the slides were dried in a 60℃ oven. After dewaxing, the sections were stained with safranin-fast green for approximately 2 minutes, washed with water, and dried at 60℃. The sections were cleared in fresh xylene for 5 minutes and mounted with neutral resin. Images were acquired under an optical microscope using CaseViewer software, and leaf thickness was measured at 30x magnification. The correlation analysis function in GraphPad Prism 5 was used to analyze the correlation between average leaf thickness and spider mite infestation level, and the two-tailed p-value was calculated.

[0097] ZmSMR3 Subcellular localization Using Jing92 cDNA as a template, PCR amplification was performed. ZmSMR3The full-length coding sequence of the fusion protein was obtained and then cloned into the pCAMBIAsuper1300-GFP expression vector. To observe the localization of the fusion protein in living cells, the recombinant plasmid was introduced into maize mesophyll protoplasts using a polyethylene glycol-mediated transfection method. For precise localization, co-transfection with Golgi apparatus and endoplasmic reticulum markers tagged with mCherry was performed. After transfection, protoplasts were cultured at 28 °C for 16–24 hours to achieve stable expression. Protoplasts exhibiting stable fluorescence were observed under a confocal laser scanning microscope (Stellaris 5, Leica Microsystems). GFP and mCherry signals were acquired using a standard filter set, followed by image channel merging and signal analysis (Fu et al., 2023).

[0098] Transmission electron microscopy observation The tissue block (approximately 1 mm) 3 Immediately fix with 2.5% glutaraldehyde for 24 hours. After fixation, wash with phosphate-buffered saline (PBS) for 6 hours, then fix with 1% osmium tetroxide for 2 hours. Subsequently, dehydrate using a gradient of ethanol: 30% ethanol for 10 minutes, 50% ethanol for 10 minutes, 70% ethanol (containing uranium acetate) for 3 hours, 80% ethanol for 10 minutes, 95% ethanol for 15 minutes, and twice with 100% ethanol (50 minutes each time), followed immediately by immersion in propylene oxide for 30 minutes. Place the sample in a 1:1 mixture of propylene oxide and epoxy resin for 2 hours, then in pure epoxy resin for 3 hours, and then embed in fresh pure epoxy resin and polymerize at 72°C for 24 hours. Cut the embedded blocks into 70 nm thick ultrathin sections using an ultramicrotome (UC-7, Leica). After lead staining, the sections were observed using a transmission electron microscope (JEM1400, Japan).

[0099] Candidate gene overexpression and functional analysis For candidate genes, the pCAMBIA3300 vector was modified to clone the CDS sequence of the target gene into the pCAMBIA3300 vector backbone. The resulting vector was used for transient transformation of maize protoplasts to verify transformation efficiency. ZmSMR3 Overexpression vector map such as Figure 18 As shown.

[0100] The vector plasmid was transferred into Agrobacterium EHA105 via electroporation. Using freshly peeled corn embryos (approximately 1 mm in diameter), each embryo was placed in a 2 ml plastic centrifuge tube containing 1.8 mL of suspension. Approximately 150 immature embryos were processed within 30 minutes. The suspension was then removed, leaving the corn embryos in the tube. 1.0 ml of Agrobacterium suspension was added to the remaining embryos, and the tubes were incubated for 5 minutes. The embryos in the centrifuge tubes were then resuspended and transferred to a co-culture medium. Excess Agrobacterium suspension was removed from the surface using a pipette, and the tubes were co-cultured in the dark at 23 °C for 3 days. After co-culture, the embryos were transferred to resting medium and incubated in the dark at 28 °C for 6 days. They were then transferred to selection medium containing diammonium phosphate for two weeks of selection culture, followed by two weeks of selection culture on a fresh selection medium. Resistant callus tissue was transferred to differentiation medium and cultured at 25 °C, 5000 lx, under light for 3 weeks. The differentiated seedlings were then transferred to rooting medium and cultured at 25 °C, 5000 lx, under light until rooting occurred. The seedlings were then transferred to small pots for further growth, and after a certain growth stage, transplanted into a greenhouse. Seeds were harvested 3-4 months later. To identify spider mite resistance, overexpressing plants were cultured in an insect-free greenhouse to stage V18, inoculated with spider mites, and the spider mite damage level was recorded according to a previous method (Di et al., 2024).

[0101] Example 3-1. High mite resistance of Jing92 and its response to spider mite infestation. Two improved strains of Huangzao IV – Jing 92 and Jing 2416 – exhibited drastically different resistance to spider mites. Ten days after spider mite infestation, the leaves of Jing 92 remained relatively green with limited damage; while the leaves of Jing 2416 showed yellowing and wilting, accompanied by severe necrotic spots. Figure 14 Image A in the figure represents a representative image of leaves infected with spider mites 7 days after inoculation for both strains. Scale bar indicates 2 cm. Phenotypic quantification results also showed that the leaf damage rating and spider mite count of Jing 92 were significantly lower than those of Jing 2416. Figure 14 B and C in the figure represent the statistics of spider mite damage level and spider mite number in "Jing 92" and "Jing 2416" cultivated in the greenhouse 7 days after inoculation. The data are expressed as the average value ± standard error of 30 individual plants.

[0102] Example 3-2. Correlation between maize spider mite resistance and leaf thickness Spider mite infestation was observed in all RIL lines, but the severity varied. Figure 15In Figure A, A represents representative images of resistant and susceptible plants from 318 recurrent populations generated from the cross between "Jing 92" and "Jing 2416" 15 days after spider mite infection. Overall, in resistant lines, the damaged area on the third leaf remained green, with lesion coverage less than 30%; while in susceptible lines, the leaves showed withered edges, and lesion coverage exceeded 70%. Consistent with our previous results, "Jing 92" exhibited relatively strong spider mite resistance in both growing seasons. Figure 15 In the figure, B represents the spider mite infestation level of the two parents and the recurrent population under two different growing conditions. The histogram represents the mean ± standard error of 20 two-parent lines and 318 recurrent populations (each population has three to five replicates). The other parent, Jing 2416, is more susceptible to the disease. The average resistance rating of the RIL population is between that of the two parents, indicating that there is an additive effect on spider mite resistance.

[0103] Based on our previous finding that the leaf thickness of Jing 92 was significantly greater than that of the susceptible inbred line (Di et al., 2024), we measured the leaf thickness at the ear position to assess whether this physiological trait plays a role in spider mite resistance throughout the RIL population. The measured leaf thickness varied considerably, ranging from 141.7 to 324 µm. Figure 15 In the image, C represents a representative cross-section of a leaf from a recurrent population under an optical microscope at 30x magnification (upper and lower parts: representative cross-sections of relatively thick and thin leaves), and it shows a significant negative correlation with the severity of spider mite infestation. (Pearson) r The value is -0.63 ( Figure 15 In the equation, D represents the correlation and linear regression analysis between the average leaf thickness in the recurrent population and the level of spider mite damage. The two-tailed p-value of the Pearson correlation coefficient is less than 0.0001, indicating that it is significant. This indicates that the thicker the leaf, the stronger the resistance to spider mites.

[0104] Example 3-3. Major QTL localization and candidate gene cloning Using the genetic map constructed for the Jing 2416 × Jing 92RIL population (Wang et al., 2024), a total of 6 QTLs associated with spider mite resistance were identified over two growing seasons (Table 3-1 and 3-2). Figure 16 In this context, A represents the chromosomal location of a QTL (Quality of Tract Resistance to Spider Mites) and its corresponding log-likelihood ratio (LOD) value, found throughout the genome. This location was determined using a genetic map constructed for the RIL population, and the LOD cutoff value was determined through 1000 permutations at a significance level of 0.05. A QTL- qSMR3-1 It was located on chromosome 3 (Chr3) and detected in both growing seasons, with phenotypic explained rates (%PVE) of 14.3% and 5.3% in Hainan and Beijing, respectively. Other QTLs ( Figure 16A) in were respectively named qSMR3-2, qSMR4 qSMR5, qSMR6 and qSMR9 , and were all detected under only one growth condition. Their contribution rates to phenotypic variation were 4.5%, 4.7%, 3.4%, 5.7% and 3.2% respectively (Table 3-1 and Figure 16 A) in, indicating that these are minor-effect QTLs. qSMR3-1 As the major-effect QTL identified in Hainan, it covered a 15-Mb genomic region and contained 237 genes.

[0105] Table 3-1. QTLs regulating spider mite resistance identified using RIL constructed by the cross of Jing92×Jing2416 a QTLs were named according to the general rules (McCouch et al., 1997), starting with the lowercase letter q, followed by the trait name and the chromosome number where the locus is located; when multiple loci are mapped on the same chromosome, they are distinguished by -1, -2.

[0106] b % PVE: Phenotypic contribution rate explained by a single quantitative trait locus.

[0107] c Additive effect: A positive value represents that the Jing92 allele has a synergistic effect, and a negative value indicates that the Jing2416 allele can enhance the target trait effect To further finely map the qSMR3-1 QTL interval and identify candidate genes controlling spider mite resistance, an F2 population was constructed by crossing two phenotypically extreme recombinant inbred lines (RILs) - 007 (highly resistant) and 009 (highly susceptible). These two inbred lines differed only in qSMR3-1 haplotypes, and had the same haplotypes for all other minor-effect QTLs. We developed 16 SNP markers using the polymorphisms between Jing92 and Jing2416 within the 15-Mb target region. By genotyping and phenotyping 2,000 F2 individuals, the QTL was finally narrowed down to a 1-Mb interval between marker IDs 17 and 19 ( Figure 16 B in, B is a schematic diagram of the 3rd and 16th SNP markers between the two parents within the 15-M region of the major QTL qSMR3-1. Fine mapping was performed using an F2 population consisting of 2,000 individuals from highly resistant and susceptible RILs. The red bar represents the 1-M region containing the spider mite resistance locus). In the Jing92 genome, this interval contains 10 annotated genes (Wang et al., 2023). Among them, ZmSMR3 the gene had significantly higher basal and induced transcriptional levels in Jing92 than in Jing2416 ( Figure 16 The C in the text refers to the concentration of C in the leaves of the two parent plants before and after spider mite infection. ZmSMR3 The relative expression is represented by the mean ± standard error for each biological replicate (5 individual plants per replicate). and (These represent p < 0.05 and p < 0.001, respectively).

[0108] Furthermore, this gene was upregulated 6-fold in Jing92 after spider mite infection, while it was only upregulated 1.5-fold in Jing2416. Sequence analysis of this gene showed that, compared with Jing2416, Jing92 had a 5bp deletion 2bp upstream of the ATG region and a 6bp insertion in the coding region. Figure 16 In the D, D represents the ratio of the two parents. ZmSMR3 A schematic diagram of natural allelic variation is shown below, illustrating the 364-base pair deletion in the promoter region of the Jing92 gene and the non-synonymous single nucleotide polymorphisms in its coding sequence. Therefore, this gene was identified as a candidate gene conferring resistance to spider mites and named [Genius name missing]. SPIDER MITE RESISTANCE 3 (SMR3) . ZmSMR3 Annotated as an exostosinlike gene, also known as a glycosyltransferase (Zhang et al., 2023; Zhong & Ye, 2003), it suggests a possible involvement in the biosynthesis of cell wall polysaccharides. To assess its biological function, we transiently expressed it in maize protoplasts. ZmSMR3 -eGFP fusion protein was used to determine its subcellular localization. The results showed that... ZmSMR3 Located in the endoplasmic reticulum and Golgi apparatus ( Figure 16 In the context of E, E represents transient overexpression. ZmSMR3 - Subcellular localization of the GFP fusion protein in maize leaf protoplasts. Endoplasmic reticulum and Golgi apparatus markers bound to mCherry were co-transformed, and GFP and mCherry fluorescence are shown in green and purple, respectively. Images were taken approximately 16 hours after transfection (bars represent 5 micrometers), consistent with its expected function as a glycosyltransferase involved in the biosynthesis of cell wall polysaccharides.

[0109] Examples 3-4. ZmSMR3 Functional validation in terms of resistance to spider mites ZmSMR3To investigate the anti-mite function, a CRISPR-Cas9 knockout (KO) line was created in the context of Jing92. Five target sites were designed and transiently transformed into Jing92 protoplasts to evaluate editing efficiency. The two target sites with the highest editing efficiency were selected, cloned into a binary vector, and introduced into Jing92, resulting in two independent mutant alleles that led to frameshift, premature termination, and protein truncation. These were named... ZmSMR3 cr Compared to the Beijing 92, ZmSMR3 cr Homozygous mutants showed significantly increased susceptibility to spider mite infestation, exhibiting more severe leaf damage and chlorophyll loss, OE- ZmSMR3 Overexpression plants are the opposite (e.g. Figure 17 In the A and C, A refers to Jing92 overexpression (OE-) 7 days after spider mite infection in the seedling stage. ZmSMR3 ) and knockout system ( ZmSMR3 cr (A representative leaf of the plant, scale bar 2 cm, where C is the number of spider mites on each plant), confirming... ZmSMR3 It is a key contributing factor to the resistance of spider mites under the genetic background of Jing 92. Figure 17 D and E in the text are ZmSMR3 The association analysis diagram between the T / C SNP genotype and resistance to maize spider mites is shown in Figure D, where D is a scatter plot of KASP genotyping, clearly distinguishing the three genotypes TT, TC, and CC; E shows the correlation between genotype and spider mite damage severity, demonstrating... ZmSMR3 The T allele at the locus positively regulates maize spider mite resistance.

[0110] In addition, overexpression in mite-susceptible strains ZmSMR3 Subsequently, the overexpressing plants exhibited stronger resistance to spider mites, with reduced leaf damage and chlorophyll loss. Consistent with this phenotype, the spider mite damage level of the overexpressing lines was significantly increased (e.g., Figure 17 In the figure, B represents the spider mite infestation level for each plant, and the number of spider mites per plant is significantly reduced (e.g., ...). Figure 17 In the figure, C represents the number of spider mites per plant. ZmSMR3 It is a key contributing factor to spider mite resistance.

[0111] ZmSMR3 Nucleotide sequence of the gene (SEQ ID NO. 5) (blue background indicates CDS region) ZmSMR3 The amino acid sequence (SEQ ID NO. 6) of gene (Zm00001eb143230) is as follows: MGGQQNALHQLVSFILGASAAAVLLFFLTTATSGARFTGISSWANGTTGFDDDAPVQAAPATRANHADAKGAAAEQEDELQRLLRAVADEDRTVIMTSVNEAWAAQDSLLDLFLESFRSGERIAHFVDHLLVVALDGGALERCRAVHPHCYLLPTAAARNLSGEKVFMSKDYIDLVWS KVRLQQRILELGYNFLFTDVDILWFRNPFERMSVAAHMVTSSDFYFGDPYSPMNLPNTGFLYAKSSRRTVGAFEAWHAAREAFPGKHEQQVLNEIKVELLATRGLRIQFLDTEHNAGFCNNTRDFNTLYTMHANCCVGLGAKLHDLGNLLQEWRAYRQMDDEERARGPVRWKVPGICIH The above embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and are not intended to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or improve the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features, within the scope of the technology disclosed in the present invention; and these modifications, changes, or substitutions 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, and should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. ZmSMR1 The application of genes in improving the resistance of corn to spider mites is characterized by, The ZmSMR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the application includes upregulating the gene. ZmSMR1 Gene expression in maize can enhance its resistance to spider mites.

2. ZmSMR1 The application of genes in genetic breeding to improve the resistance of corn to spider mites, the aforementioned ZmSMR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the application includes upregulating the gene. ZmSMR1 Gene expression in maize can enhance its resistance to spider mites.

3. ZmSMR1 The application of genes in germplasm improvement to enhance the resistance of maize to spider mites, the aforementioned ZmSMR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the application includes upregulating the gene. ZmSMR1 Gene expression in maize can enhance its resistance to spider mites.

4. ZmSMR1 The application of the gene in the production of transgenic maize resistant to spider mites, the aforementioned ZmSMR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the application includes upregulating the gene. ZmSMR1 Gene expression in maize can enhance its resistance to spider mites.

5. ZmSMR1 The application of genes in regulating maize cell wall structure is characterized by, By raising ZmSMR1 Gene expression in maize increases the content of pectin and hemicellulose in maize cell walls, remodels cell wall structure, and thickens leaves and cell walls; ZmSMR1 The gene encodes the GT47 family of glycosyltransferases, which ZmSMR1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. A breeding method for maize with high resistance to spider mites, characterized in that, This includes the following steps: by adjusting upwards ZmSMR1 The expression of the gene in maize enhances the maize's resistance to spider mites, wherein... ZmSMR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

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