Corn ear rot resistance related molecular marker, related detection primer pair and application

Abstract

The present application provides a corn smut resistance related molecular marker which is closely linked to corn smut resistance, and the nucleotide sequence is shown as SEQ ID NO:1, and the C / T base mutation exists at the 101th base from the 5' end of the nucleotide sequence shown as SEQ ID NO:1. The present application also provides a primer pair for detecting the corn smut resistance related molecular marker and a kit for detecting the corn smut resistance. The corn smut resistance related molecular marker of the present application can detect the corn smut resistance at the seedling stage, so that the screening of corns resistant to smut can be realized at the seedling stage, and the breeding process of corns resistant to smut can be accelerated.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and maize genetics and breeding technology, and particularly to the field of molecular marker technology related to maize ear rot resistance, specifically to a molecular marker related to maize ear rot resistance, related detection primer pairs, and their applications. Background Technology

[0002] Corn is an important food, feed, and energy crop worldwide. Ear rot is a common fungal disease in corn production caused by various pathogens, with *Fusarium graminearum* and *Fusarium verticillatum* being the two dominant pathogens in my country. Ear rot not only significantly reduces the commercial quality of corn kernels but also causes severe yield losses, seriously threatening corn production. Furthermore, the pathogens produce various toxins such as deoxynivalenol and zearalenone, posing significant risks to food and feed safety and seriously threatening human and animal health.

[0003] Resistance to ear rot in maize is a complex quantitative trait, influenced by genotype, environment, and the interaction between genotype and environment. Using traditional field selection breeding methods for genetic improvement of maize ear rot resistance is not only inaccurate but also time-consuming and costly.

[0004] With the development of molecular technology and the widespread application of molecular markers, new tools have been provided for improving resistance to maize ear rot. Among the many types of molecular markers, KASP molecular markers are widely used due to their high accuracy, low detection cost, and rapid result analysis. KASP molecular markers rely on allele-specific amplification and fluorescence signal determination to achieve accurate typing of target single nucleotide polymorphisms, making them particularly suitable for rapid screening in large-scale populations.

[0005] Therefore, it is hoped that a molecular marker related to maize ear rot resistance can be developed, which can detect maize ear rot resistance at the seedling stage, thereby enabling the screening of ear rot-resistant maize at the seedling stage, which is of great significance for the rapid breeding of new maize varieties resistant to ear rot. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, one object of the present invention is to provide a molecular marker related to maize ear rot resistance, which can detect maize ear rot resistance at the seedling stage, thereby enabling the screening of ear rot resistant maize at the seedling stage, thus accelerating the breeding process of ear rot resistant maize, and is suitable for large-scale promotion and application.

[0007] Another objective of this invention is to provide an application of molecular markers related to maize ear rot resistance, which can be used to detect maize ear rot resistance, detect maize ear rot resistance genotypes, or screen ear rot resistant maize breeding materials. This allows for the detection of maize ear rot resistance at the seedling stage and the screening of ear rot resistant maize at the seedling stage, thereby accelerating the ear rot resistant maize breeding process and making it suitable for large-scale promotion and application.

[0008] Another objective of this invention is to provide a primer pair for detecting molecular markers related to maize ear rot resistance. This primer pair can detect molecular markers related to maize ear rot resistance, thereby enabling the detection of maize ear rot resistance at the seedling stage and the screening of ear rot-resistant maize at the seedling stage. This accelerates the breeding process of ear rot-resistant maize and is suitable for large-scale promotion and application.

[0009] Another objective of this invention is to provide a kit for detecting maize ear rot resistance, which can detect molecular markers related to maize ear rot resistance, thereby enabling the detection of maize ear rot resistance at the seedling stage and the screening of ear rot-resistant maize at the seedling stage, thus accelerating the breeding process of ear rot-resistant maize and making it suitable for large-scale promotion and application.

[0010] Another objective of this invention is to provide a primer pair for detecting molecular markers related to maize ear rot resistance or a kit for detecting maize ear rot resistance. This kit can be used to detect molecular markers related to maize ear rot resistance, thereby enabling the detection of maize ear rot resistance at the seedling stage and the screening of ear rot-resistant maize at the seedling stage. This accelerates the breeding process of ear rot-resistant maize and is suitable for large-scale application.

[0011] Another objective of this invention is to provide a primer pair for detecting molecular markers related to maize ear rot resistance or a kit for detecting maize ear rot resistance. This kit can be used to detect maize ear rot resistance, detect maize ear rot resistance genotypes, or screen for ear rot-resistant maize. This allows for the detection of maize ear rot resistance at the seedling stage and the screening of ear rot-resistant maize at the seedling stage, thereby accelerating the breeding process of ear rot-resistant maize and making it suitable for large-scale application.

[0012] To achieve the above objectives, in a first aspect of the present invention, a molecular marker for resistance to corn ear rot is provided, characterized in that the molecular marker for resistance to corn ear rot is closely linked to resistance to corn ear rot, and the nucleotide sequence of the molecular marker for resistance to corn ear rot is shown in SEQ ID NO: 1, wherein the nucleotide sequence shown in SEQ ID NO: 1 has a C / T base mutation at position 101 from the 5' end.

[0013] In a second aspect of the invention, the application of the above-mentioned molecular markers related to maize ear rot resistance is provided in detecting maize ear rot resistance, detecting maize ear rot resistance genotypes, or screening for ear rot resistant maize.

[0014] In a third aspect of the invention, primer pairs are provided for detecting molecular markers related to maize ear rot resistance, wherein the molecular markers related to maize ear rot resistance are those described above.

[0015] Preferably, the primer pair includes the first forward primer shown in SEQ ID NO: 2, the second forward primer shown in SEQ ID NO: 3, and the reverse primer shown in SEQ ID NO: 4.

[0016] More preferably, the 5' end of the first forward primer and the 5' end of the second forward primer are respectively connected to different fluorescent adapter sequences.

[0017] Furthermore, the 5' end of the first forward primer is connected to a FAM fluorescent adapter sequence, and the 5' end of the second forward primer is connected to a HEX fluorescent adapter sequence.

[0018] In a fourth aspect of the invention, a kit for detecting resistance to ear rot in maize is provided, characterized in that it includes the primer pairs described above.

[0019] In a fifth aspect of the invention, the application of the primer pairs described above for detecting molecular markers related to maize ear rot resistance or the kit described above for detecting maize ear rot resistance is provided in the detection of the aforementioned molecular markers related to maize ear rot resistance.

[0020] In a sixth aspect of the invention, the application of the primer pairs described above for detecting molecular markers related to maize ear rot resistance or the kit described above for detecting maize ear rot resistance is provided in detecting maize ear rot resistance, detecting maize ear rot resistance genotypes, or screening for ear rot resistant maize.

[0021] Preferably, the DNA of the maize material to be tested is amplified by PCR using the primer pair, and the amplification product is analyzed. If the position of the 101st base of the amplification product corresponding to the above-mentioned molecular marker related to maize ear rot resistance is C, then the maize material to be tested has resistance to maize ear rot; if the position of the 101st base of the amplification product corresponding to the above-mentioned molecular marker related to maize ear rot resistance is T, then the maize material to be tested does not have resistance to maize ear rot.

[0022] The main beneficial effects of this invention are:

[0023] 1. The molecular marker for maize ear rot resistance of the present invention is closely linked to maize ear rot resistance. The nucleotide sequence is shown in SEQ ID NO: 1. The nucleotide sequence shown in SEQ ID NO: 1 has a C / T base mutation at position 101 from the 5' end. Therefore, it can detect maize ear rot resistance at the seedling stage, thereby enabling the screening of ear rot resistant maize at the seedling stage, thus accelerating the breeding process of ear rot resistant maize, and is suitable for large-scale promotion and application.

[0024] 2. The application of the above-mentioned molecular markers related to maize ear rot resistance in the present invention in detecting maize ear rot resistance, detecting maize ear rot resistance genotypes, or screening ear rot resistant maize, therefore, can be used to detect maize ear rot resistance, detect maize ear rot resistance genotypes, or screen ear rot resistant maize breeding materials, thereby enabling the detection of maize ear rot resistance at the seedling stage and the screening of ear rot resistant maize at the seedling stage, thus accelerating the ear rot resistant maize breeding process and making it suitable for large-scale promotion and application.

[0025] 3. The primer pair of the present invention for detecting molecular markers related to maize ear rot resistance, wherein the molecular markers related to maize ear rot resistance are the aforementioned molecular markers related to maize ear rot resistance, thus it can detect molecular markers related to maize ear rot resistance, thereby enabling the detection of maize ear rot resistance at the seedling stage, enabling the screening of ear rot-resistant maize at the seedling stage, thereby accelerating the breeding process of ear rot-resistant maize, and is suitable for large-scale promotion and application.

[0026] 4. The kit for detecting maize ear rot resistance of the present invention includes the primer pairs mentioned above. Therefore, it can detect molecular markers related to maize ear rot resistance, thereby enabling the detection of maize ear rot resistance at the seedling stage and the screening of ear rot-resistant maize at the seedling stage, thus accelerating the breeding process of ear rot-resistant maize and making it suitable for large-scale promotion and application.

[0027] 5. The application of the primer pairs for detecting molecular markers related to maize ear rot resistance or the kit for detecting maize ear rot resistance described above in the detection of the aforementioned molecular markers related to maize ear rot resistance can be used to detect molecular markers related to maize ear rot resistance, thereby enabling the detection of maize ear rot resistance at the seedling stage and the screening of ear rot-resistant maize at the seedling stage, thus accelerating the breeding process of ear rot-resistant maize and making it suitable for large-scale promotion and application.

[0028] 6. The primer pairs for detecting molecular markers related to maize ear rot resistance described above, or the kits for detecting maize ear rot resistance described above, can be used to detect maize ear rot resistance, detect maize ear rot resistance genotypes, or screen for maize resistant to ear rot. Therefore, they can be used to detect maize ear rot resistance, detect maize ear rot resistance genotypes, or screen for maize resistant to ear rot. This allows for the detection of maize ear rot resistance at the seedling stage and the screening of maize resistant to ear rot at the seedling stage, thereby accelerating the breeding process of maize resistant to ear rot and making it suitable for large-scale promotion and application.

[0029] These and other objects, features and advantages of the present invention will be fully apparent from the following detailed description, drawings and claims, and may be achieved by the means, products and combinations thereof specifically pointed out in the appended claims. Attached Figure Description

[0030] Figure 1 This is a map showing the genetic loci mapping of maize ear rot resistance in Example 1.

[0031] Figure 2 The image shows the genotyping results of the K10 molecular marker in 195 maize inbred lines in Example 3. In the image, T:T indicates that the genotype of the detection site is T / T homozygous, C:C indicates that the genotype of the detection site is C / C homozygous, C:T indicates that the genotype of the detection site is C / T heterozygous, ? indicates that the genotype of the detection site is unknown, and NTC indicates the blank control.

[0032] Figure 3 The image shows a violin plot of the disease index distribution of 189 maize inbred lines that were identified as homozygous genotypes using the K10 molecular marker in Example 3. The C / C genotype at the detection site is a homozygous resistant genotype, and the T / T genotype at the detection site is a homozygous susceptible genotype. Detailed Implementation

[0033] To develop molecular markers related to maize ear rot resistance, the inventors, based on F2 populations derived from maize varieties resistant to ear rot and susceptible to ear rot, combined population genotype data and ear rot resistance phenotypic data, identified a stable major genetic locus for maize ear rot resistance through linkage analysis. Based on this stable major genetic locus, molecular markers closely linked to it were developed, aiming to provide technical support for the genetic improvement of maize ear rot resistance, thus completing this invention.

[0034] This invention first provides a molecular marker for resistance to corn ear rot, which is closely linked to resistance to corn ear rot. The nucleotide sequence of the molecular marker for resistance to corn ear rot is shown in SEQ ID NO: 1. The nucleotide sequence shown in SEQ ID NO: 1 has a C / T base mutation at position 101 starting from the 5' end.

[0035] The present invention also provides the application of the above-mentioned molecular markers related to maize ear rot resistance in detecting maize ear rot resistance, detecting maize ear rot resistance genotypes, or screening maize resistant to ear rot.

[0036] The present invention also provides primer pairs for detecting molecular markers related to maize ear rot resistance, wherein the molecular markers related to maize ear rot resistance are the aforementioned molecular markers related to maize ear rot resistance.

[0037] The primer pair may have any suitable nucleotide sequence, and the primer pair includes the first forward primer shown in SEQ ID NO: 2, the second forward primer shown in SEQ ID NO: 3, and the reverse primer shown in SEQ ID NO: 4.

[0038] For easier detection, it is preferable that the 5' ends of the first forward primer and the second forward primer are respectively connected to different fluorescent adapter sequences.

[0039] The different fluorescent adapter sequences connected to the 5' ends of the first forward primer and the second forward primer can be determined as needed. Furthermore, the 5' end of the first forward primer is connected to the FAM fluorescent adapter sequence, and the 5' end of the second forward primer is connected to the HEX fluorescent adapter sequence.

[0040] This invention also provides a kit for detecting resistance to maize ear rot, comprising the primer pairs described above. This invention does not have specific requirements for primer concentration, but a concentration of 10 µM is recommended.

[0041] The present invention also provides the application of the primer pairs for detecting molecular markers related to maize ear rot resistance or the kit for detecting maize ear rot resistance in detecting the aforementioned molecular markers related to maize ear rot resistance.

[0042] The present invention also provides the application of the primer pairs for detecting molecular markers related to maize ear rot resistance or the kits for detecting maize ear rot resistance in detecting maize ear rot resistance, detecting maize ear rot resistance genotypes, or screening for ear rot resistant maize.

[0043] The application can be performed using any suitable steps. Preferably, the primer pair is used to perform PCR amplification on the DNA of the maize material to be tested, and the amplification product is analyzed. If the position of the 101st base of the amplification product corresponding to the above-mentioned molecular marker for maize ear rot resistance is C, then the maize material to be tested has maize ear rot resistance. If the position of the 101st base of the amplification product corresponding to the above-mentioned molecular marker for maize ear rot resistance is T, then the maize material to be tested does not have maize ear rot resistance.

[0044] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions as described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or as recommended by the manufacturer. The version number of the maize reference genome used in this invention is Zm-B73-REFERENCE-GRAMENE-4.0 (https: / / www.maizegdb.org / genome / assembly / Zm-B73-REFERENCE-GRAMENE-4.0).

[0045] Example 1: Obtaining the major genetic locus qGER10.03 for regulating maize ear rot resistance

[0046] (1) Test materials: The F2 population (including 168 F2 plants) was developed using the resistant inbred line SUW and the susceptible inbred line HCL645 as parents, and the F2 population was developed by self-pollination of the 168 F2 plants. 2:3 The pedigree serves as a population for locating genetic loci for resistance to maize ear rot.

[0047] All of the above materials were provided free of charge by the Jiangsu Yangtze River Agricultural Science Research Institute, which also provides these materials to the public free of charge.

[0048] (2) Genetic map construction: DNA was extracted from SUW, HCL645, and 168 F2 single plants using the CTAB method, and the quality and concentration of DNA were detected by 1% agarose gel electrophoresis. Shijiazhuang Borui Biotechnology Co., Ltd. was commissioned to analyze the genotypes of SUW, HCL645, and 168 F2 single plants using the GenoBaits® 10K liquid chromatography-mass spectrometry (LC-MS). Initially, sites homozygous for both SUW and HCL645 and exhibiting polymorphism between them were screened. Then, polymorphic markers with a deletion rate greater than 20% and severe segregation were filtered out. Finally, 2099 SNP molecular markers with polymorphism and co-segregation between parents were obtained. The genetic distance between SNP molecular markers was calculated using the Kosambi method in JoinMap 4.0 software. The total length of the genetic map was 1861 cM.

[0049] (3) Resistance identification: The F2 plants developed by self-pollination of the parents SUW and HCL645 and 168 F2 individual plants were tested. 2:3 The family was planted in Nantong, Jiangsu Province in the spring of 2023 and in Jining, Shandong Province in the summer. The field trial used a randomized block design with two replicates, single-row plots, 5 m long, 20 plants per row, and 0.6 m spacing. Ten days after silking, artificial inoculation was performed. First, an incision was made in the upper, middle, and lower parts of the first ear of corn using an awl, puncturing 1-2 kernels without reaching the cob. Then, 200 μL of Fusarium graminearum inoculum solution (1×10⁻⁶) was injected into each incision using a continuous syringe. 6 (Spores / mL), spray with water to keep moist. After the corn matures, harvest and sun-dry naturally. Classify the affected area into 7 levels based on the percentage of ear area affected: Level 1: No disease; Level 2: 1%–3% of ear area affected; Level 3: 4%–10% of ear area affected; Level 4: 11%–25% of ear area affected; Level 5: 26%–50% of ear area affected; Level 6: 51%–75% of ear area affected; Level 7: 76%–100% of ear area affected.

[0050] (4) Linkage analysis: Combining population phenotypic data and the constructed genetic map, the genetic loci regulating maize ear rot resistance were detected using the composite interval mapping method in Windows QTLCartographer 2.5 software. At the P < 0.05 level, the LOD value was determined through 1000 permutations.

[0051] (5) Obtaining qGER10.03: Results of linkage analysis (see...) Figure 1As shown in the figure, at the LOD > 3.5 level, a total of 7 genetic loci regulating maize ear rot resistance were detected, located on maize chromosomes 1, 2, 5, 6, 7, and 10. Among them, the genetic locus located on chromosome 10 was detected in both the Nantong and Jining experimental sites, explaining 13%–17% of the phenotypic variation. It is a stable major-effect resistance locus, and this locus is named qGER10.03.

[0052] Example 2: Development of molecular markers related to maize ear rot resistance

[0053] For the nucleotide sequence within 100 bp before and after the peak SNP marker in the qGER10.03 region, the KASP molecular marker K10 was developed according to primer design principles. Its primer sequences are: K10-F1 (sequence shown in SEQ ID NO: 5 + sequence shown in SEQ ID NO: 2), K10-F2 (sequence shown in SEQ ID NO: 6 + sequence shown in SEQ ID NO: 3), and K10-R (sequence shown in SEQ ID NO: 4).

[0054] The method for detecting ear rot resistance in maize K10 using the above-mentioned KASP primer set is as follows:

[0055] (1) Extract genomic DNA from the maize sample to be tested.

[0056] (2) Preparation of the reaction system. A commercially available KASP typing kit can be used. The reaction system is 10 µL, including 1 µL DNA, 0.2 µL K10-F1, 0.2 µL K10-F2, 0.6 µL K10-R, 5 µL 2×KASP Mix, and 3 µL ddH2O. In the reaction system, the concentration of DNA is 30 ng / µL, and the concentrations of K10-F1, K10-F2, and K10-R are all 10 µM.

[0057] (3) Add 6 µL of liquid paraffin to the reaction system (to prevent sample evaporation), and then perform PCR amplification.

[0058] The reaction program was as follows: 95 ℃ for 10 min; 95 ℃ for 20 sec, 61~55 ℃ for 1 min, 72 ℃ for 1 min, with the annealing temperature decreasing by 0.6 ℃ per cycle until reaching 55 ℃, for 10 cycles; 95 ℃ for 20 sec, 55 ℃ for 40 sec, 72 ℃ for 1 min, for 30 cycles.

[0059] (4) After completing step (3), the signal is read on the Douglas Scientific Array Tape platform. If only blue is displayed, the detection site is the C / C genotype, which is a homozygous type of ear rot resistance, and the corresponding maize material is determined to be resistant to ear rot. If only red is displayed, the detection site is the T / T genotype, which is a homozygous type of ear rot susceptibility, and the corresponding maize material is determined to be susceptible to ear rot. If both blue and red are displayed, the detection site is the C / T genotype, which is a heterozygous type of ear rot resistance, and the corresponding maize material is determined to have moderate resistance to ear rot.

[0060] Example 3: Application of KASP molecular marker K10 in the selection of maize ear rot resistance

[0061] Using the primer set and detection method designed in Example 2, the genotypes and genetic effects of the K10 molecular marker in 195 maize inbred lines (provided free of charge by the Jiangsu Yangtze River Agricultural Science Research Institute, which also provides this material free of charge to the public) were identified. The ear rot resistance phenotype data of the 195 maize inbred lines were the best linear unbiased predictors for five environments, cited from the paper published in Theoretical Applied Genetics in 2024 (Zhou et al. 2024. Genome-wide association study and molecular marker development for susceptibility to Gibberella ear rot in maize. Theoretical Applied Genetics, 137: 222).

[0062] The results showed that among the 195 maize inbred lines, 92 were homozygous for the C / C genotype with an average ear rot disease index of 4.09; 97 were homozygous for the T / T genotype with an average ear rot disease index of 4.29; 2 were heterozygous for the C / T genotype; and 4 had no detectable genotype. The difference in ear rot disease index between the C / C and T / T genotype inbred lines was highly significant (P-value = 7.97e-3). This indicates that the K10 genotype cosegregates in maize inbred lines and has a stable and reliable genetic effect, which can effectively predict maize ear rot resistance.

[0063] Therefore, this invention identifies a stable major genetic locus for ear rot resistance, qGER10.03, on chromosome 10 of maize through linkage analysis. Based on a SNP locus within the qGER10.03 region, a KASP molecular marker, named K10, was developed. K10 is located at position 26414445 on chromosome 10 of the maize reference genome Zm-B73-REFERENCE-GRAMENE-4.0. K10 exhibits a C / T base mutation. The nucleotide sequence of 100 bp before and after K10 is shown in SEQ ID NO.1. When the base at this locus is C (C / C genotype), the maize material can be determined to be resistant to ear rot; when the base at this locus is T (T / T genotype), the maize material can be determined to be susceptible to ear rot.

[0064] Based on this SNP site, a primer set for the KASP molecular marker was designed. The primer set includes two forward primers and one reverse primer. The 5' ends of the two forward primers are connected to different fluorescent adapter sequences, such as the FAM fluorescent adapter sequence (shown in SEQ ID NO: 5) and the HEX fluorescent adapter sequence (shown in SEQ ID NO: 6).

[0065] This invention also discloses the application of the aforementioned molecular markers and primer sets in marker-assisted breeding related to maize ear rot resistance. In other words, the molecular markers and primer sets of this invention can be used in future marker-assisted breeding to detect the genotype of the K10 molecular marker developed in this invention by extracting DNA from maize kernels or seedling leaves, thereby predicting ear rot resistance in maize materials. Taking FAM and HEX fluorescent adapter sequences as examples, if the PCR product of the sample only detects the FAM fluorescent adapter sequence, the detection site is the C / C genotype, and it is determined to be a homozygous type resistant to ear rot; if the PCR product of the sample only detects the HEX fluorescent adapter sequence, the detection site is the T / T genotype, and it is determined to be a homozygous type susceptible to ear rot; if both the FAM fluorescent adapter sequence and the HEX fluorescent adapter sequence are detected simultaneously, the detection site is the C / T genotype, and it is determined to be a heterozygous type resistant to ear rot.

[0066] The present invention has the following advantages:

[0067] (1) The inventors of this invention screened out a stable major genetic locus for maize ear rot resistance, qGER10.03, which can explain more than 10% of the phenotypic variation and has high breeding application value.

[0068] (2) Based on a SNP site in the qGER10.03 region, the KASP molecular marker developed in this invention can quickly and accurately predict maize ear rot resistance, and is suitable for high-throughput screening of large-scale breeding populations, which is beneficial to improving breeding efficiency.

[0069] In summary, the molecular markers related to maize ear rot resistance of the present invention can detect maize ear rot resistance at the seedling stage, thereby enabling the screening of ear rot-resistant maize at the seedling stage, thus accelerating the breeding process of ear rot-resistant maize, and making it suitable for large-scale promotion and application.

[0070] Therefore, it is evident that the objective of this invention has been fully and effectively achieved. The function and structural principles of this invention have been demonstrated and explained in the embodiments. Any modifications can be made to the implementation methods without departing from these principles. Therefore, this invention includes all modified embodiments based on the spirit and scope of the claims.

Claims

1. A molecular marker associated with resistance to maize ear rot, characterized in that, The molecular markers related to resistance to corn ear rot are closely linked to resistance to corn ear rot. The nucleotide sequences of the molecular markers related to resistance to corn ear rot are shown in SEQ ID NO:

1. The nucleotide sequence shown in SEQ ID NO: 1 has a C / T base mutation at position 101 starting from the 5' end.

2. The application of the molecular markers related to maize ear rot resistance as described in claim 1 in detecting maize ear rot resistance, detecting maize ear rot resistance genotypes, or screening maize resistant to ear rot.

3. Primer pairs for detecting molecular markers related to maize ear rot resistance, wherein the molecular markers related to maize ear rot resistance are those described in claim 1.

4. The primer pair according to claim 3, characterized in that, The primer pair includes the first forward primer shown in SEQ ID NO: 2, the second forward primer shown in SEQ ID NO: 3, and the reverse primer shown in SEQ ID NO:

4.

5. The primer pair according to claim 4, characterized in that, The 5' ends of the first forward primer and the second forward primer are respectively connected to different fluorescent adapter sequences.

6. The primer pair according to claim 5, characterized in that, The 5' end of the first forward primer is connected to the FAM fluorescent adapter sequence, and the 5' end of the second forward primer is connected to the HEX fluorescent adapter sequence.

7. A kit for detecting resistance to ear rot in maize, characterized in that, Includes the primer pair according to claim 3.

8. The application of the primer pair for detecting maize ear rot resistance-related molecular markers according to claim 3 or the kit for detecting maize ear rot resistance according to claim 7 in detecting the maize ear rot resistance-related molecular markers according to claim 1.

9. The primer pair for detecting molecular markers related to maize ear rot resistance according to claim 3 or the kit for detecting maize ear rot resistance according to claim 7, used in detecting maize ear rot resistance, detecting maize ear rot resistance genotypes, or screening for maize resistant to ear rot.

10. The application according to claim 9, characterized in that, The primers were used to perform PCR amplification on the DNA of the maize material to be tested. The amplification products were analyzed. If the position of the 101st base of the amplification product corresponding to the molecular marker for maize ear rot resistance according to claim 1 is C, then the maize material to be tested has resistance to maize ear rot. If the position of the 101st base of the molecular marker related to maize ear rot resistance according to claim 1 is T, then the maize material to be tested does not have resistance to maize ear rot.

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