A product for detecting SNP sites in a corn genome, and application of SNP sites in a corn genome in identifying a southern corn blight resistance trait

Abstract

This invention discloses a product for detecting SNP loci in the maize genome and the application of SNP loci in the identification of resistance to southern maize rust, relating to the field of plant molecular breeding technology. This invention identified 52 SNP loci significantly associated with resistance to southern maize rust through genome-wide association analysis. These SNP loci significantly associated with resistance can be used to identify or assist in the identification of resistance to southern maize rust. Furthermore, this invention also predicted a rust-related gene through functional annotation and expression pattern analysis, providing a favorable technical foundation for subsequent aggregation of rust-resistant loci to achieve broad-spectrum resistance to southern maize rust in maize breeding.

Description

Technical Field

[0001] This invention relates to the field of plant molecular breeding technology, and more specifically, to a product for detecting SNP sites in the maize genome and the application of SNP sites in the maize genome in identifying resistance to southern maize rust. Background Technology

[0002] Maize is a major food crop and a primary source of feed and energy for light industry. However, diseases can reduce maize yield and quality, and in severe cases, lead to complete crop failure. The most economical and effective method for controlling diseases is to develop maize lines carrying resistance genes, with the identification and cloning of these genes being crucial steps in achieving this goal. Although 11 dominant maize resistance genes (Rpp1 to Rpp11) and 8 major maize resistance QTLs (RppC, RppCML470, RppD, RppM, RppP25, RppQ, RppS, and RppS313) have been reported, only RppC and RppK have been cloned (Ding et al., 2022; Chen et al., 2022). Discovering and cloning new loci for resistance to southern maize rust and achieving broad-spectrum resistance through aggregation is of great significance.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a product for detecting SNP sites in the maize genome and the application of SNP sites in the maize genome in identifying resistance to southern maize rust, thereby solving the above-mentioned technical problems.

[0005] This invention is implemented as follows:

[0006] In a first aspect, the present invention provides the application of a substance for detecting the genotype of SNP sites in the maize genome in identifying or assisting in the identification of resistance to southern maize rust, wherein the SNP sites are selected from at least one of the following SNP site numbers:

[0007]

[0008]

[0009] Secondly, the present invention also provides a product for detecting SNP sites in the maize genome, which is used to detect at least one of the above-mentioned SNP sites, and the product is a reagent, kit or chip.

[0010] Thirdly, the present invention also provides the application of at least one SNP site or the above-mentioned product in genome-wide association analysis of resistance to southern maize rust.

[0011] Fourthly, the present invention also provides the application of at least one SNP site or the above-mentioned product in maize-assisted breeding or maize breeding resistant to southern maize rust.

[0012] Fifthly, the present invention also provides the application of at least one SNP site in the preparation of a detection chip or detection kit for SNP sites against southern maize rust.

[0013] Sixthly, the present invention also provides the application of gene Zm00001 d037626 in plant rust resistance. Gene Zm00001 d037626 encodes serine-threonine protein phosphatase 2a; and the physical location of the gene is 132302357bp-132311438bp; the physical location of the gene is determined based on the B73 whole genome V4.0 version.

[0014] Seventhly, the present invention also provides the application of a vector including gene Zm00001 d037626 in plant rust resistance, wherein gene Zm00001d037626 encodes serine-threonine protein phosphatase 2a; and the physical location of the gene is 132302357bp-132311438bp; the physical location of the gene is determined based on B73 whole genome version V4.0.

[0015] The present invention has the following beneficial effects:

[0016] This invention utilizes an association analysis population constructed from 388 superior inbred lines for planting. The plants are naturally susceptible to disease, and resistance is assessed at maturity based on the susceptibility of the plants. Genotyping of the 388 inbred lines was performed using a maize 50K high-density SNP microarray, obtaining 37,283 SNP markers covering the entire genome to determine plant genotypes. Genome-wide association analysis identified 52 SNP loci significantly associated with resistance to southern maize rust. These SNP loci can be used to identify or assist in identifying resistance to southern maize rust. By determining the genotype at these SNP loci in the test sample, the resistance to southern maize rust can be identified or its identification can be aided.

[0017] Furthermore, based on the reference genome sequence of the inbred line B73, this invention identified 168 genes potentially associated with resistance to southern maize rust within a 100kb range of 52 SNPs. One gene, Zm00001d037626, potentially related to resistance to southern maize rust, was screened out. This gene encodes serine-threonine protein phosphatase 2a. This protein catalyzes the phosphorylation of amino acids such as serine and threonine. This enzyme is an important component of amino acid metabolism and signal transduction pathways in plants, participating in various biological processes such as growth and development, stress defense, and metabolic regulation. Therefore, the screened gene shows promising application potential for plant rust resistance. This invention not only uncovers new regulatory sites for resistance to southern maize rust but also provides a possibility for the subsequent aggregation of rust resistance sites to achieve broad-spectrum resistance. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 These are the GWAS results; where A is the QQ plot and B is the Manhattan plot.

[0020] Figure 2 This is a chain imbalance diagram;

[0021] Figure 3 The graph shows the bioinformatics analysis results of candidate genes; where A represents the genes contained within the 100Kb interval of the most significant point, and B represents the expression pattern of candidate genes. Detailed Implementation

[0022] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0023] Definitions:

[0024] SNP (Single Nucleotide Polymorphism): Single nucleotide polymorphism refers to DNA sequence polymorphism caused by a single nucleotide variation at the genomic level.

[0025] MAF (Minor Allele Frequency): The minimum allele frequency, usually refers to the frequency of an uncommon allele at a specific locus in a particular population. For example, with genotypes TT, TC, and CC, if the frequency of C is 0.36 and the frequency of T is 0.64, then allele C is the minimum allele frequency, and the MAF is 0.36. When an SNP has three alleles, the second most common allele frequency is defined as the MAF.

[0026] (See https: / / www.ncbi.nlm.nih.gov / projects / SNP / docs / rs_attributes.html#gmaf). For example, if an SNP locus has three alleles: A, C, and G, with a frequency of 0.5 for A, 0.4 for C, and 0.1 for G, then the MAF (Magnitude of Effectiveness) is 0.4. In general population analyses, an MAF > 0.05 is required because a smaller MAF yields less usable information, reduces statistical power, and increases the likelihood of false positives.

[0027] In a first aspect, the present invention provides the application of a substance for detecting the genotype of SNP sites in the maize genome in identifying or assisting in the identification of resistance to southern maize rust, wherein the SNP sites are selected from at least one of the following SNP site numbers:

[0028]

[0029]

[0030]

[0031] The SNP loci screened in this invention are closely related to rust resistance phenotypes. By detecting the genotypes of these SNP loci in maize, resistance to southern maize rust can be identified or further identified. These SNP loci are located on nine of the ten chromosomes in the maize genome, mainly concentrated in the bin regions 1.04, 2.08, 3.09, 5.00, and 6.05. The ten most significant SNPs explain 4.5%-7.4% of the total phenotypic variation, improving the accuracy of related research applications. The combinations of these SNP loci provide a wealth of useful information and have significant utilization value. Based on these SNP loci, it is helpful to further explore broad-spectrum rust-resistant inbred lines or composites with multiple rust-resistant loci, and apply them to the breeding of rust-resistant maize varieties.

[0032] In a preferred embodiment of the present invention, the SNP site is selected from at least one of the following SNP site numbers:

[0033]

[0034]

[0035] In a preferred embodiment of the present invention, the SNP sites are selected from at least one of the following SNP site numbers: SNP-1, SNP-5, and SNP-21 (based on B73 whole genome version V4.0). These three SNP sites are sites with significant rust resistance. These three SNP sites are in linkage disequilibrium, located within the same LD BLOCK, and the 100Kb intervals surrounding these three sites contain three identical genes: Zm00001 d037626, Zm00001 d037627, and Zm00001 d037628.

[0036] In a preferred embodiment of the present invention, the application includes: the application includes the following genotypes for resistance to southern maize rust at SNP-1 to SNP-52, in that order:

[0037]

[0038]

[0039]

[0040] If the genotype of at least one SNP locus among SNP-1 to SNP-52 in the maize sample being tested matches the genotype for resistance to southern maize rust, then the maize sample is identified as having resistance to southern maize rust. For example, the genotype for resistance to southern maize rust corresponding to SNP-10 is G. At least one SNP locus among the aforementioned SNP-1 to SNP-52 includes, but is not limited to, two SNP loci, three SNP loci, four SNP loci, five SNP loci, eight SNP loci, ten SNP loci, twenty SNP loci, thirty SNP loci, forty SNP loci, fifty SNP loci, or fifty-two SNP loci, sequentially satisfying the genotypes shown above, then the maize sample is identified as having resistance to southern maize rust.

[0041] In a preferred embodiment of the present invention, if the genotypes of at least one SNP locus at the above-mentioned physical location of SNP-1, SNP-5 and SNP-21 in the maize sample to be tested satisfy the C, A and G genotypes respectively, then the maize sample to be tested is identified as having resistance to southern maize rust.

[0042] In a preferred embodiment of the present invention, if the genotypes of SNP-1, SNP-5, and SNP-21 at the aforementioned physical locations in the maize sample to be tested satisfy the C, A, and G genotypes respectively, then the tested maize sample is identified as having resistance to southern maize rust. Through verification experiments, the present invention has found that the above three SNP loci can effectively genotype maize resistant to southern maize rust and are significant rust-resistant loci.

[0043] In one optional embodiment, primers for detecting at least one of the aforementioned SNP loci are designed, and then the genotype of the corresponding SNP locus is detected. The resistance to southern maize rust is determined based on the genotype. In other embodiments, the resistance to southern maize rust is determined by comprehensively detecting the genotype of a combination of multiple SNP loci in the sample to be tested.

[0044] In a preferred embodiment of the present invention, the substance used to detect the genotype of SNP sites in the maize genome is selected from at least one of the following: primers, probes, reagents including primers and / or probes, kits including primers and / or probes, and chips including primers and / or probes. The 5' end of the probe is modified with a fluorescent reporter group, for example, and the 3' end of the probe is modified with a fluorescent quencher group. The fluorescent reporter group is HEX, FAM, TET, CF532, JOE, TAMRA, ROX, CY3, CY5, CY5.5, Texas Red, NED, Alexa Flour, or VIC, and the quencher group is MGB, TAMRA, BHQ1, BHQ2, BHQ3, or QSY.

[0045] To rapidly detect SNP sites, those skilled in the art can couple primers and probes onto microcarrier substrate materials (such as chips) via chemical coupling or other methods to achieve the detection of one or more SNP sites. For example, primers and probes can be embedded within the detection chamber or cavity of the chip.

[0046] Secondly, the present invention also provides a product for detecting SNP sites in the maize genome, which is used to detect at least one of the aforementioned SNP sites. The product is a reagent, kit, or chip. The reagent includes, but is not limited to, solid (e.g., lyophilized powder), liquid, or semi-solid.

[0047] Thirdly, the present invention also provides the application of at least one SNP locus or the aforementioned product in genome-wide association analysis of resistance to southern maize rust. For example, at least one of the aforementioned SNP loci can be used for fine analysis of resistance to southern maize rust, or the aforementioned SNP locus can be combined with existing SNP loci for genome-wide association analysis of resistance to southern maize rust.

[0048] Fourthly, this invention also provides the application of at least one SNP site or the aforementioned product in maize-assisted breeding or in the breeding of maize resistant to southern maize rust. The discovery of the aforementioned SNP sites helps to identify broad-spectrum rust-resistant inbred lines or composites with multiple rust-resistant sites, which can then be applied to the breeding of rust-resistant maize varieties.

[0049] Fifthly, the present invention also provides the application of at least one SNP site in the preparation of a detection chip or detection kit for SNP sites against southern maize rust.

[0050] By preparing SNP chips and combining them with targeted genotyping technology using liquid-phase probe hybridization, standardized and automated detection and analysis can be easily achieved.

[0051] Sixthly, this invention also provides the application of a gene in plant rust resistance. The gene Zm00001d037626 encodes serine-threonine protein phosphatase 2a, and its physical location is 132302357bp-132311438bp; the physical location of the gene is determined based on the B73 whole genome version V4.0. This protein is responsible for catalyzing the phosphorylation of amino acids such as serine and threonine. This enzyme is an important component of amino acid metabolism and signal transduction pathways in plants, participating in various biological processes such as growth and development, stress defense, and metabolic regulation. For example, the article "Pathogen protein modularity enables elaborate mimicry of a host phosphatase" published in *Cell* found that *Phytophthora infestans* effector proteins can recruit serine-threonine protein phosphatase, and RNA silencing inhibitors can bind to this protein, thereby enhancing plant sensitivity and accelerating disease spread.

[0052] In a preferred embodiment of the present invention, the nucleotide sequence of the gene is shown in SEQ ID NO.1;

[0053] In a preferred embodiment of the present invention, the application includes any of the following application methods:

[0054] (1) Introduce genes into target plant cells;

[0055] (2) Transform the target plant with a vector containing the gene;

[0056] (3) Introduce recombinant bacteria or recombinant cells into the target plant. The recombinant bacteria or recombinant cells contain genes.

[0057] Seventhly, the present invention also provides the application of a vector including gene Zm00001d037626 in plant rust resistance, wherein gene Zm00001d037626 encodes serine-threonine protein phosphatase 2a; the physical location of the gene is 132302357bp-132311438bp; the physical location of the gene is determined based on B73 whole genome version V4.0.

[0058] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0059] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0060] This invention constructs an associated population using 388 inbred lines widely planted in China, and cultivates them in Xinxiang, Henan Province. Phenotypic results are obtained by strictly classifying disease-susceptible plants under natural conditions. Genotypes are then obtained by scanning a 50K high-density SNP chip in maize. Using a mixed linear model, rust-resistant loci are located throughout the genome. The aim is to explore broad-spectrum rust-resistant inbred lines or aggregates with multiple rust-resistant loci, and apply them to the breeding of rust-resistant maize varieties.

[0061] Example 1

[0062] This embodiment provides a screening method for 52 sites that are significantly associated with resistance to southern maize rust.

[0063] (1) In the summer of 2023, 388 core inbred lines from various maize-producing areas across the country were selected and planted in Xinxiang, Henan Province, during the peak season in the Huang-Huai-Hai maize-producing region. Each inbred line was planted in one row, with a row length of 5 meters and a total of 21 plants. In October 2023, before the maize harvest, a statistical survey was conducted on their resistance to southern maize rust. All 388 maize inbred lines showed full disease development and were generally severely affected. Among them, 10% had resistance levels of 1 and 3, and 67% had resistance levels of 7 and 9 (the values ​​range from 1 to 9, representing the severity of southern maize rust; the smaller the value, the stronger the resistance). In the same plot, the improved line of the rust-resistant inbred line K22 had a resistance level of 3, while the susceptible inbred line ZHENG58 had a resistance level of 9.

[0064] (2) Using the high-density gene chip Maize 50K (maize 50K SNP chip genotyping platform), the genotypes of the 388 inbred lines mentioned above were identified in accordance with the standard procedure of Illumina Infinium gene chip detection, and 37,283 SNP markers covering the entire genome were obtained.

[0065] (3) Using TASSEL5.0 software, the minimum allele frequency (MAF) was set to 0.05 for quality control. Principal component analysis (PCA) and kinship matrix were used to obtain covariate data for GWAS analysis. Then, the PCA results were used as population structure covariates for general linear model (GLM) analysis.

[0066] The quantile-quantile plot showed that at the initial loci, such as before -log10 = 3, the p-values ​​were significantly higher than those of a uniform distribution, indicating false positives and suggesting that the GLM model was not suitable. Subsequently, a mixed linear model (MLM) combining population structure and kinship was attempted for analysis. The quantile-quantile plot showed that at the initial loci, before -log10 = 3, the p-values ​​were consistent with those of a uniform distribution; however, after the p-values ​​exceeded 3, the loci began to deviate from the straight line and became upward-sloping. Figure 1 Figure A in the diagram shows that the MLM model is suitable. (See Manhattan diagram for reference.) Figure 1 Figure B in the diagram.

[0067] (4) Using a mixed linear model (Q+K), GWAS identified 52 loci significantly associated with resistance to southern maize rust. The following mixed linear model (Q+K) was used to analyze the association between the markers and the trait of resistance to southern maize rust:

[0068] y = Xβ + Zμ + ε, where y is the phenotypic observation, β is the fixed effects vector including genetic markers and population structure, μ is the random additive genetic effects vector for individuals / lines, X and Z are the design matrices for unknown fixed and random effects, respectively, ε is the residual effect, μ and ε follow normal distributions, K is the genomic kinship matrix, is the additive genetic variance, and is the residual variance. Multiple tests with correction factors are used to determine the significance level P of the loci. This significance level P reflects the degree of association between the marker and phenotypic variation; the smaller the P value, the higher the association between the marker and phenotypic variation. Different significance thresholds are set to screen out significant loci for association analysis.

[0069] The statistical analysis results of the 52 sites are shown in the table below:

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] Example 2

[0076] Gene annotation was performed on the 52 SNPs screened in Example 1.

[0077] GWAS using a mixed linear model identified 52 SNPs significantly associated with resistance to southern maize rust. These 52 SNPs were distributed across chromosomes 1, 2, 3, 4, 5, 6, 8, 9, and 10, primarily concentrated in the bin regions 1.04, 2.08, 3.09, 5.00, and 6.05. The 10 SNPs with the highest significance explained 4.5%–7.4% of the total phenotypic variation.

[0078] The 52 significant rust-resistant sites encompass 168 genes within a 100Kb range. According to the reference genome B73 annotation, these genes may be involved in catalytic activity, binding activity, metabolic processes, cellular processes, biological regulation, stress response, and developmental processes. Among them, there are many genes related to serine-threonine proteins, sugar transporters, cytochromes, and glutathione metabolism.

[0079] The most significant site among the 52 sites, SNP-1 (P = 0.0000011669), is in linkage disequilibrium with two other significant anti-rust sites, SNP-5 (P = 0.00006021) and SNP-21 (P = 0.00033054), and they are located within the same LD block. Figure 2 These three loci contain three identical genes within a 100kb interval: Zm00001d037626 (132302357bp-132311438bp), Zm00001d037627 (132317697bp-132319277bp), and Zm00001d037628 (132320661bp-132325847bp). Figure 3(See Figure A in the diagram). Among them, genes Zm00001d037627 and Zm00001d037628 are not expressed in leaves, which is inconsistent with the location of southern maize rust disease. Therefore, it is believed that gene Zm00001d037626 may be related to resistance to southern maize rust. Simultaneously, this gene is predicted to encode serine-threonine protein phosphatase 2a, which is responsible for catalyzing the phosphorylation of amino acids such as serine and threonine. This enzyme is an important component of amino acid metabolism and signal transduction pathways in plants, participating in various biological processes such as growth and development, stress defense, and metabolic regulation. Figure 3 (Figure B in the diagram).

[0080] For example, the article "Pathogen protein modularity enables elaborate mimicry of a host phosphatase" published in *Cell* found that *Phytophthora infestans* effector proteins can recruit serine-threonine protein phosphatases, and RNA silencing inhibitors can bind to these proteins, thereby enhancing plant susceptibility and accelerating disease spread. Therefore, the gene Zm00001d037626 shows promising application potential in breeding varieties resistant to southern maize rust.

[0081] Example 3

[0082] This study references the phenotypes of approximately 500 natural populations resistant to southern maize rust published in *Nature Communications* by the maize team at Huazhong Agricultural University, entitled "Cloningsouthern corn rust resistant gene RppK and its cognate gene AvrRppK from Puccinia polysora". This example analyzes the genotypes of SNP-1, SNP-5, and SNP-21 in each of the approximately 500 rust-resistant natural populations from that study. Based on the genotypes (C, A, and G) at these three SNP loci, the approximately 500 rust-resistant natural populations were categorized into rust-resistant and non-rust-resistant types. A t-test was performed on the rust-resistant samples and SNP loci, revealing that inbred lines with genotypes C, A, and G for SNP-1, SNP-5, and SNP-21 showed significantly stronger resistance to southern maize rust, with a p-value of 0.0102 (statistical results are shown in the table below).

[0083]

[0084] These findings indicate that SNP-1, SNP-5, and SNP-21 are key loci for identifying plant resistance to southern maize rust, and the nearby gene Zm00001 d037626 is a key gene. Based on this invention, the SNP-1, SNP-5, and SNP-21 associated with rust-related genes can be used to genotype this natural population.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of substances for detecting the genotype of SNP locus combinations in the maize genome in assisting in the identification of maize resistance to southern maize rust, characterized in that, The SNP locus combination is a combination of the following SNP locus numbers: ， The physical location of the SNP loci was determined based on the B73 whole genome version V4.

0.

2. The application according to claim 1, characterized in that, The substance used to detect the genotype of SNP site combinations in the maize genome is: primers.

3. The application according to claim 1, characterized in that, The substance used to detect the genotype of SNP site combinations in the maize genome is a probe.

4. The application according to claim 1, characterized in that, The substances used to detect the genotype of SNP site combinations in the maize genome are reagents including primers and / or probes.

5. The application according to claim 1, characterized in that, The material used to detect the genotype of SNP site combinations in the maize genome is a kit containing primers and / or probes.

6. The application according to claim 1, characterized in that, The material used to detect the genotype of SNP site combinations in the maize genome is a chip containing probes.

7. The application of the substance for detecting the genotype of SNP locus combinations in the maize genome as described in any one of claims 1-6 in the breeding of maize resistant to southern maize rust.

8. The application of the substance for detecting the genotype of SNP site combinations in the maize genome as described in any one of claims 1, 3 and 6 in the preparation of a maize resistance detection chip for southern maize rust.

9. The application of the substance for detecting the genotype of SNP site combinations in the maize genome as described in any one of claims 1-6 in the preparation of a maize resistance test kit for southern maize rust.

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