A major resistance qtl and its linked molecular markers for goss' wilt caused by f. pseudograminearum in maize

Abstract

The application discloses a main-effect QTL of corn ear rot (FER) resistance caused by Fusarium verticilloides and a molecular marker linked to the main-effect QTL. The main-effect QTL of corn ear rot resistance is located in a physical interval of about 1.73 Mb on a 7.01-7.02 bins of a corn chromosome 7, and the main-effect QTL can explain a phenotypic variation rate of 11.76-18.02%. Three SNP molecular markers are closely linked to the main-effect QTL, and the three SNP molecular markers are Zyf788, Zyf792 and Zyf7105. The main-effect QTL of corn ear rot resistance and the molecular marker linked to the main-effect QTL are applied to screening or identifying corn varieties with ear rot resistance. The application helps to overcome the characteristics of high cost and long time in identifying ear rot resistance in corn disease breeding work, and greatly accelerates the cultivation and industrialization process of new corn varieties with high ear rot resistance in China.

Description

Technical Field

[0001] This invention belongs to the field of crop genetics and breeding, and designs a major QTL for maize ear rot resistance and its linked SNP molecular marker. Background Technology

[0002] Corn is the world's largest food crop, and diseases have always been a major limiting factor affecting the increase of corn yield and the improvement of quality. Corn ear rot is a fungal disease that occurs widely and seriously all over the world. It is also a major disease in various corn-producing areas in my country. In normal years, the incidence of corn ear rot is 10%-20%, and in severe years it can reach 30%-40%. The incidence of susceptible varieties is as high as about 50%, and the severity of the disease is increasing year by year. In the latest draft of the corn approval standard, ear rot has become a disease that will be rejected in Northeast, Huanghuai and Southwest regions. Corn ear rot has three main harms: (1) it causes the ears to rot, which directly leads to a reduction in yield; (2) some pathogens can also produce a large number of harmful toxins, which seriously threaten the health and safety of humans and animals; (3) the occurrence of corn ear rot leads to an increase in moldy corn kernels. When the moldy kernel content exceeds the national standard (≤2%), the corn will not be able to be sold, resulting in a large amount of infected corn accumulating in the farmers' storage, which in turn causes a series of problems such as corn purchase safety, circulation safety, storage safety and quality safety. According to surveys, the moldy kernel content caused by ear rot in corn in Jilin Province was 3.4% and 2.9% in 2014 and 2015, respectively, resulting in significant economic losses for corn farmers. Furthermore, direct harvesting of corn kernels has become a trend in corn breeding. However, during direct harvesting, it is impossible to remove ears infected with ear rot. Therefore, the continuous advancement of mechanization will place even stricter demands on the ear rot resistance of corn varieties.

[0003] Because most inbred lines currently used in maize breeding lack resistance to ear rot, the resistance situation for maize ear rot in my country is very serious and urgently needs improvement. Screening resistant inbred lines and breeding resistant varieties are the most fundamental and effective measures for controlling and preventing maize ear rot. Therefore, it is crucial to locate quantitative trait loci for maize ear rot resistance, discover corresponding resistance genes, and utilize molecular marker-assisted breeding and biotechnology breeding to improve maize resistance to ear rot. Summary of the Invention

[0004] The purpose of this invention is to provide a novel major QTL site for maize with high resistance to ear rot, along with its associated molecular markers and applications.

[0005] This invention claims protection for a major QTL-qRfer10 associated with resistance to maize ear rot, the major QTL site being located on maize chromosome 7, bins 7.01-7.02.

[0006] This invention also claims protection for molecular markers linked to the major QTL-qRfer10 associated with resistance to maize ear rot, namely A1, A2 and / or A3.

[0007] A1, molecular marker Zyf788, wherein the molecular marker Zyf788 is a SNP site in the maize genome, the molecular marker Zyf788 is located at nucleotide 151 of SEQ ID No. 1 on chromosome 7 of the maize genome, and it is either A or G;

[0008] A2, molecular marker Zyf792, wherein the molecular marker Zyf792 is a SNP site in the maize genome, the molecular marker Zyf792 is located at nucleotide 151 of SEQ ID No.2 on chromosome 7 of the maize genome, and it is G or T;

[0009] A3, molecular marker Zyf7105, wherein the molecular marker Zyf7105 is a SNP site in the maize genome, located at nucleotide 151 of SEQ ID No. 3 on chromosome 7 of the maize genome, and is either C or G.

[0010] The molecular markers Zyf788, Zyf792 and / or Zyf7105 are used in any of the following:

[0011] 1) Application in identifying or assisting in the identification of maize ear rot resistance;

[0012] 2) Applications in maize breeding;

[0013] 3) Application in screening or identifying maize varieties resistant to ear rot;

[0014] 4) Application in screening or identifying the main effective QTL-qRfer10 for resistance to ear rot in maize.

[0015] Application of substances that detect the polymorphism or genotype of molecular marker Zyf788 in the maize genome, substances that detect the polymorphism or genotype of molecular marker Zyf792 in the maize genome, and / or substances that detect the polymorphism or genotype of molecular marker Zyf7105 in the maize genome in the preparation of products for identifying or assisting in the identification of maize ear rot resistance.

[0016] The substance used to detect the polymorphism or genotype of the molecular marker Zyf788 in the maize genome is a primer pair that amplifies maize genomic DNA fragments including Zyf788.

[0017] The substance used to detect the polymorphism or genotype of the molecular marker Zyf792 in the maize genome is a primer pair that amplifies maize genomic DNA fragments including Zyf792;

[0018] The substance used to detect the polymorphism or genotype of the molecular marker Zyf7105 in the maize genome is a primer pair that amplifies maize genomic DNA fragments including Zyf7105.

[0019] A method for identifying or assisting in the identification of maize ear rot resistance: The method involves detecting the genotypes of molecular markers Zyf788, Zyf792, and / or Zyf7105 in the genome of the plant to be tested. Plants with the genotype GG of molecular marker Zyf788 exhibit lower resistance to maize ear rot than plants with the genotypes AG and AA of molecular marker Zyf788; plants with the genotype TT of molecular marker Zyf792 exhibit lower resistance to maize ear rot than plants with the genotypes GT and GG of molecular marker Zyf792; and plants with the genotype GG of molecular marker Zyf7105 exhibit lower resistance to maize ear rot than plants with the genotypes GC and GG of molecular marker Zyf7105.

[0020] The application of the substances for detecting the polymorphism or genotype of molecular marker Zyf788 in the maize genome, the substances for detecting the polymorphism or genotype of molecular marker Zyf792 in the maize genome, and / or the substances for detecting the polymorphism or genotype of molecular marker Zyf7105 in the maize genome in maize breeding.

[0021] A kit for identifying or assisting in the identification of maize ear rot resistance, the kit comprising the substances described in claim 3 for detecting the polymorphism or genotype of molecular marker Zyf788 in the maize genome, the substances for detecting the polymorphism or genotype of molecular marker Zyf792 in the maize genome, and / or the substances for detecting the polymorphism or genotype of molecular marker Zyf7105 in the maize genome.

[0022] The application of the kit in identifying or assisting in the identification of maize ear rot resistance.

[0023] The corn ear rot mentioned is caused by Fusarium moniliforme.

[0024] The beneficial effects of this invention are as follows: A genetic linkage map is constructed using the F2 population of a cross between the highly resistant maize ear rot inbred line Dan598 and the highly susceptible maize ear rot inbred line ZW18. Self-pollination of the F2 population yields the F1 generation. 2:3 The family was inoculated with Fusarium verticillatum 12 days after silking. Disease incidence was assessed in maize ears at maturity, and the results were recorded for each F1 generation. 2:3The average resistance rating of the families represents the corresponding resistance rating of individual F2 plants. Then, combining F2 population genetic linkage data and resistance data, a major QTL for high resistance to ear rot in maize, qRfer10, was obtained through QTL mapping, located on maize chromosome 7, bins 7.01-7.02. Subsequently, QTL-qRfer10 was finely mapped to a physical region of approximately 1.73 Mb (refer to B73_RefGen_v4) through backcrossing of exchanged plants for verification. QTL-qRfer10 explains 11.76-18.02% of the phenotypic variation. It is closely linked to three SNP markers: Zyf788, Zyf792, and Zyf7105. This QTL can be introduced into maize varieties susceptible to ear rot using marker-assisted selection, thereby simultaneously achieving genetic improvement of maize ear rot resistance. Furthermore, it provides important theoretical basis and genetic resources for cloning resistance genes and molecular breeding of maize for disease resistance. Attached Figure Description

[0025] Figure 1 The polymorphic sites of the SNP molecular marker Zyf788 in Example 2 were detected.

[0026] Figure 2 The polymorphic sites of the SNP molecular marker Zyf792 in Example 2 were detected.

[0027] Figure 3 The genetic linkage and phenotypic data of the F2 population in Example 4 were plotted using WinQTLCart software with composite intervals. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] The highly resistant maize ear rot inbred line Dan 598 described in the following examples, Zhang Yan, Zhang Ye, et al. Identification of resistance of 44 maize inbred lines to Fusarium ear rot. Journal of Plant Genetic Resources, 2019, 20(2):276-283, can be obtained from the Maize Research Institute of Jilin Academy of Agricultural Sciences.

[0031] The highly susceptible maize ear rot inbred line ZW18 described in the following examples, Wen Jing, Shen Yanqi, et al. QTL mapping of maize resistance to Fusarium verticillatum ear rot based on image analysis. Chinese Journal of Agricultural Science, 2021, 54(13):2724-2736, is available to the public from the Maize Research Institute of Jilin Academy of Agricultural Sciences.

[0032] The Fusarium verticilloides described in the following examples, Zhang Yan, Zhang Ye, et al. Resistance identification of 44 maize inbred lines to Fusarium ear rot. Journal of Plant Genetic Resources, 2019, 20(2):276-283, can be obtained from the laboratory of Professor Xu Mingliang of China Agricultural University.

[0033] The inoculation method of Fusarium verticilloides in the following embodiment includes the following steps:

[0034] 1. Cultivation of the pathogen: Inoculate *Fusarium graminearum* onto PDA agar plates and incubate at 28°C for approximately one week until the mycelium has fully colonized the plate. Store at 4°C for later use. Boil 40g of mung beans in an appropriate amount of water for 20 minutes, filter, add 20g of sucrose and 1g of KH₂PO₄, and bring the volume to 1L. Autoclave at 121°C for 30 minutes, cool, and store at 4°C for later use. Cut half of the cultured agar plate into 0.25cm pieces. 2 Small pieces were placed in 500 mL of 5% mung bean decoction liquid culture medium and cultured at 28℃ with shaking at 150 rpm for 2 days. The mycelia were filtered, and the spores were collected and stored at 4℃ for later use. One day before inoculation, a solution of 5 × 10⁻⁶ mung bean decoction liquid culture medium was prepared using sterile distilled water. 6 A spore suspension of spores / mL was prepared. Tween-80, a surfactant, was added to the spore suspension at a concentration of 2 μl / mL. -1 Mix well.

[0035] 2. Artificial inoculation: Based on past experimental experience, the optimal inoculation time is 12 days after silking. Inject the prepared spore suspension into the middle of the corn ear using a syringe. Inject 2 mL of spores into each ear. After inoculation, gently pinch the wound with your fingers to prevent the spores from flowing out.

[0036] In the following examples, the criteria for judging maize ear rot resistance were as follows: a field disease survey was conducted during the maize maturity period (late September in Gongzhuling, Jilin Province). The husks of each inoculated maize ear were removed, and the disease status of each ear was investigated and recorded individually. The average disease severity level was calculated. Referring to the "Technical Specification for Identification of Maize Disease and Insect Resistance NY-T 1248.8-2106", resistance was divided into 5 levels: 1, 3, 5, 7, and 9, where level 1 represents high resistance and level 9 represents high susceptibility.

[0037] Table 1 Grading Criteria for Maize Ear Rot Disease

[0038]

[0039]

[0040] Example 1. Discovery of a major-effect QTL, qRfer10, for maize with high resistance to ear rot.

[0041] A genetic linkage map was constructed using the F2 population of a cross between the highly resistant maize ear rot inbred line Dan598 and the highly susceptible maize ear rot inbred line ZW18. F2 self-pollination was then used to obtain the F1 generation. 2:3 The family was inoculated with *Fusarium verticillatum* 12 days after silking. Disease incidence was assessed in maize ears at maturity, and the results were recorded for each F1 generation. 2:3 The average resistance rating of the families represents the corresponding resistance rating of individual F2 plants. QTL mapping was then performed by combining F2 population genetic linkage data and resistance data. Validation was then conducted through backcrossing of exchanging individual plants. The results showed that QTL-qRfer10 is located on maize chromosome 7, bins 7.01-7.02, with a fine mapping to a physical region of approximately 1.73 Mb (refer to B73_RefGen_v4). QTL-qRfer10 explains 11.76-18.02% of the phenotypic variation. It is closely linked to three SNP markers: Zyf788, Zyf792, and Zyf7105. Among them, the SNP molecular marker Zyf788 is located at the 8845552nd base on chromosome 7 of maize (corresponding to the 151st position in sequence 1), and the SNP functional molecular marker polymorphism is 'A / G', denoted by r; the SNP molecular marker Zyf792 is located at the 9259475th base on chromosome 7 of maize (corresponding to the 151st position in sequence 2), and the SNP functional molecular marker polymorphism is 'G / T', denoted by k; the SNP molecular marker Zyf7105 is located at the 10574604th base on chromosome 7 of maize (corresponding to the 151st position in sequence 3), and the SNP functional molecular marker polymorphism is 'C / G', denoted by s.

[0042] Example 2. SNP detection of maize ear rot resistance using molecular markers Zyf788, Zyf792 and Zyf7105

[0043] 1. Primer design

[0044] For the SNP molecular marker Zyf788, two forward-specific fluorescent primer sequences, such as SEQ ID No. 8 and SEQ ID No. 9, and a reverse universal primer sequence, such as SEQ ID No. 10, were designed.

[0045] Among them, the forward specific primer sequences (Specific-allele X and Specific-allele Y) of the SNP molecular marker Zyf788 are as shown in SEQ ID No. 4 and SEQ ID No. 5, respectively, and the fluorescent sequence tags SEQ ID No. 6 and SEQ ID No. 7 are connected to the 5' end to form the forward specific fluorescent primer sequences as shown in SEQ ID No. 8 and SEQ ID No. 9, respectively, and the reverse universal primer sequence is as shown in SEQ ID No. 10.

[0046] For the SNP molecular marker Zyf792, two forward-specific fluorescent primer sequences, such as SEQ ID No. 13 and SEQ ID No. 14, and a reverse universal primer sequence, such as SEQ ID No. 15, were designed.

[0047] Among them, the forward specific primer sequences (Specific-allele X and Specific-allele Y) of the SNP molecular marker Zyf792, such as SEQ ID No. 11 and SEQ ID No. 12, are connected to the fluorescent sequence tags SEQ ID No. 6 and SEQ ID No. 7 at the 5' end to form forward specific fluorescent primer sequences such as SEQ ID No. 13 and SEQ ID No. 14, and the reverse universal primer sequence is such as SEQ ID No. 15.

[0048] For the SNP molecular marker Zyf7105, two forward-specific fluorescent primer sequences, such as SEQ ID No. 18 and SEQ ID No. 19, and a reverse universal primer sequence, such as SEQ ID No. 20, were designed.

[0049] Among them, the forward specific primer sequences (Specific-allele X and Specific-allele Y) of the SNP molecular marker Zyf7015 are as shown in SEQ ID No.16 and SEQ ID No.17, respectively. The fluorescent sequence tags SEQ ID No.6 and SEQ ID No.7 are connected to the 5' end to form the forward specific fluorescent primer sequences as shown in SEQ ID No.18 and SEQ ID No.19, respectively, and the reverse universal primer sequence is as shown in SEQ ID No.20.

[0050] Using the above primers to verify the parent lines, the following was found:

[0051] The two forward-specific fluorescent primer sequences for the SNP molecular marker Zyf788 are shown in SEQ ID No. 8 and SEQ ID No. 9, and the reverse universal primer sequence is shown in SEQ ID No. 10. In the resistant parent Dan598, the polymorphic site 'A / G' has a base of 'A', meaning that the fluorescent signal corresponding to the fluorescent sequence tag in SEQ ID No. 8 (e.g., SEQ ID No. 6) can be detected in the resistant parent Dan598. In the susceptible parent ZW18, the polymorphic site 'A / G' has a base of 'G', meaning that the fluorescent signal corresponding to the fluorescent sequence tag in SEQ ID No. 9 (e.g., SEQ ID No. 7) can be detected in the susceptible parent ZW18. Both fluorescent signals can be detected in a mixed sample of Dan598 and ZW18.

[0052] The two forward-specific fluorescent primer sequences for the SNP molecular marker Zyf792 are shown in SEQ ID No. 13 and SEQ ID No. 14, and the reverse universal primer sequence is shown in SEQ ID No. 15. In the resistant parent Dan598, the polymorphic site 'G / T' has a base of 'G', meaning that the fluorescent signal corresponding to the fluorescent sequence tag in SEQ ID No. 13, such as SEQ ID No. 6, can be detected in the resistant parent Dan598. In the susceptible parent ZW18, the polymorphic site 'G / T' has a base of 'T', meaning that the fluorescent signal corresponding to the fluorescent sequence tag in SEQ ID No. 14, such as SEQ ID No. 7, can be detected in the susceptible parent ZW18. Both fluorescent signals can be detected in a mixed sample of Dan598 and ZW18.

[0053] Two forward-specific fluorescent primer sequences for the SNP molecular marker Zyf7105 are shown in SEQ ID No. 18 and SEQ ID No. 19, and a reverse universal primer sequence is shown in SEQ ID No. 20. In the resistant parent Dan598, the polymorphic site 'C / G' has a base of 'C', meaning that the fluorescent signal corresponding to the fluorescent sequence tag in SEQ ID No. 18, such as SEQ ID No. 6, can be detected in the resistant parent Dan598. In the susceptible parent ZW18, the polymorphic site 'C / G' has a base of 'G', meaning that the fluorescent signal corresponding to the fluorescent sequence tag in SEQ ID No. 19, such as SEQ ID No. 7, can be detected in the susceptible parent ZW18. Both fluorescent signals can be detected in a mixed sample of Dan598 and ZW18.

[0054] The molecular marker method for identifying the major-effect QTL-qRfer10 for maize high resistance to ear rot as described in this invention utilizes one or more primer pairs from the SNP molecular markers Zyf788, Zyf792, and Zyf7105 to perform fluorescence detection on the maize material sample to be identified. If the corresponding base or fluorescence is detected, it indicates that the major-effect QTL-qRfer10 for maize high resistance to ear rot is present.

[0055] 2. Construction of maize hybrid lines

[0056] The maize inbred line Dan 598, which is highly resistant to ear rot, and the inbred line ZW18, which is highly susceptible to ear rot, were used to construct an F2 population and an F2 population of size 200. 2:3 The lineage was used to locate the QTLs for maize ear rot resistance.

[0057] The maize inbred line Dan598, which is highly resistant to ear rot, was crossed with the inbred line ZW18, which is highly susceptible to ear rot. After obtaining the F1 generation, the line was self-crossed twice to obtain the F2 population and the corresponding F3 generation. 2:3 The family, with a population size of 200, was used to locate the QTL for maize ear rot resistance.

[0058] 3. F2 group and F 2:3 resistance to ear rot in maize populations

[0059] The primer combinations designed in step 1 were used to detect the F2 population and F2 population, respectively. 2:3 The polymorphism of individual plants in the lineage was studied, and the F2 generation was obtained through self-pollination of the F2 generation. 2:3 The family was inoculated with Fusarium verticillatum 12 days after silking. Disease incidence was assessed in maize ears at maturity, and the results were recorded for each F1 generation. 2:3 The average resistance rating of the family represents the corresponding resistance rating of the F2 individual plant, as shown in Tables 1 and 2.

[0060] Primer composition for detecting F2 population and F 2:3 The steps for determining the polymorphism of individual plants within a population are as follows:

[0061] 1) Genomic DNA extraction: A simplified CTAB method was used to extract genomic DNA from maize leaves. First, a small amount of leaves was placed in a mortar, and 1.2 mL of 2% CTAB extraction buffer was added. The leaves were ground into a homogenate and transferred to a 2.0 mL centrifuge tube. The sample was incubated at 65°C for 1 h, shaking 3-5 times during incubation. After cooling to room temperature, 700 μL of chloroform:isoamyl alcohol (24:1) was added and the mixture was shaken inverted for 2-3 min to mix. The sample was centrifuged at 11000 g for 10 min, and 600 μL of the supernatant was transferred to a new 1.5 mL centrifuge tube containing 600 μL of isopropanol. The sample was gently shaken for 30 sec. After DNA flocculent material appeared, the sample was centrifuged at 11000 g for 1 min, and the supernatant was discarded. 800 μL of 75% ethanol was added, and the DNA precipitate was washed twice. The sample was centrifuged at 11000 g for 30 sec, and the supernatant was discarded. The sample was dried at room temperature, and then dissolved in 100 μL of ddH2O. The sample was stored at -20°C for later use.

[0062] 2) PARMS reaction: Using the maize genomic DNA obtained in step 1) as a template, a PCR reaction is performed using fluorescent specific primers. The forward primer is the specific primer described in claim 2, with different fluorescent tag sequences connected to the 5' end, and the reverse primer is the universal primer described in claim 2.

[0063] Fluorescence detection: If only the fluorescent sequence tag attached to primer Specific-allele X, such as the one corresponding to SEQ ID No. 6, is detected, the tested corn is a corn sample resistant to corn ear rot. If only the fluorescent sequence tag attached to primer Specific-allele Y, such as the one corresponding to SEQ ID No. 7, is detected, the tested corn is determined to be a corn sample susceptible to corn ear rot. If both fluorescence signals are detected simultaneously, the tested corn is determined to be a hybrid sample of corn resistant to corn ear rot and corn susceptible to corn ear rot.

[0064] Table 1. SNP polymorphisms and maize ear rot resistance in the F2 population.

[0065]

[0066]

[0067]

[0068]

[0069] Table 2 Resistance ratings for different genotypes

[0070]

[0071] For individuals in the parental and F2 populations, the maize GenoBaits Maize10K chip developed by Shijiazhuang Borui Biotechnology Co., Ltd. was used for detection. Genotypes matching the bases of the susceptible parent ZW18 were marked as "1", those matching the bases of the resistant parent Dan598 were marked as "2", and heterozygous sites showing bands from both parents were marked as "3". After removing partial segregation markers and duplicate markers from the obtained genotype data, linkage maps were constructed using MAPMAKER 3.0. The "suggest subset" command was used to construct the framework of linkage groups, inferring possible linkage groups with LOD values ​​greater than 3.0. The Kosambi function was used to convert recombination values ​​to map distance units (cM). The "threepoint" and "order" commands were used to orient the markers on the linkage groups to obtain the genetic linkage map. Figure 3 As shown, the total genetic distance of the obtained genetic linkage map is 1862 cM, and the average genetic distance between any two adjacent markers is 4.22 cM. Among them, the linkage group length of chromosome 7 is 138 cM, and the number of markers in the linkage group is 30.

[0072] For parents and F 2：3 Families were assessed for resistance using artificial inoculation with *Fusarium verticillatum* at two locations in Jilin Province: Gongzhuling and Songyuan. Two replicates were set up at each location to obtain phenotypic data. For each F1 line... 2:3 The mean phenotype of the line represents the corresponding phenotypic value of F2. Then, combining the genetic linkage data and phenotypic data of the F2 population, the QTL for maize ear rot resistance was scanned using the composite interval plotting (CIM) method with WinQTLCart software. The threshold of LOD value was automatically generated by the software using the permutation test method, with 300 permutations and a significance level of 0.05.

[0073] The major-effect QTL-qRfer10 detected was characterized by being located on maize chromosome 7, bins 7.01-7.02. A genetic linkage map was constructed using an F2 population derived from a cross between the highly resistant maize ear rot inbred line Dan 598 and the highly susceptible maize ear rot inbred line ZW18. F2 individuals were then self-crossed to obtain F10 cells. 2:3 The family was inoculated with Fusarium verticillatum 12 days after silking. Disease incidence was assessed in maize ears at maturity, and the results were recorded for each F1 generation. 2:3The average resistance rating of the families represents the corresponding resistance rating of individual F2 plants. QTL mapping was then performed by combining F2 population genetic linkage data and resistance data. Subsequently, QTL-qRfer10 was finely mapped to a physical region of approximately 1.73 Mb (refer to B73_RefGen_v4) through backcrossing of exchanging individual plants for verification. QTL-qRfer10 explains 11.76–18.02% of the phenotypic variation. It is closely linked to three SNP markers: Zyf788, Zyf792, and Zyf7105.

[0074] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <110> Jilin Academy of Agricultural Sciences <120> A major QTL for resistance to maize ear rot caused by Fusarium oxysporum and its linked molecular markers <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 301 <212> DNA <213> Artificial Sequence <400> 1 aaatcgcctc tgcgtgctga atctgtgcag cgtgactcgt gcgaaggaca ctgcagagga 60 tcagccgaag ggatgatggg tgtatctgta gaatgtctgt atctgaatgg agtcgatgcg 120 cccttgtcgc cacatttcga cctttcaagc rgtctgatcc ggcagtgtaa agatttgcct 180 tcaatatctt ccactttgtg caccttcggc tcgcttacat gttccaaagc ttttcaaaga 240 gcaatagttt ttctttacct ctcattagca tgcttgacct agaagatcat ctaagcattg 300 a 301 <210> 2 <211> 301 <212> DNA <213> Artificial Sequence <400> 2 acaaatcaac cagacggttt ttgcaggatc tgttgcaaaa aaaaaaggcg gatatttgtt 60 tggtgggatc ccttgtttgg ttggtggacc agtacggggc tggtggtgca tcacgagatc 120 aagaccggaa actgatgtgc gcaaagagat ktctacgggc ggaaaaaagt gtctgatagc 180 aacatttcac atggttgctg atctaccatc gatcgagttc gagtcgacac tgggaatcta 240 atgcggataa ctacagaaac aactgggcgt tgactgttat taagaggata acctcaacct 300 c 301 <210> 3 <211> 301 <212> DNA <213> Artificial Sequence <400> 3 gtcaagttct tcctgctcga agttgctgcg tgggtgatgt ttgccttcct agagacatac 60 acgaggctgt tctggtcgtc ggagtccctg tactacaacc ccgcctggca cgcgatcgtc 120 tcttcttggg aaaccgtcgt ctacagcccc sttcttgatc ttgacaggtc aggagttctc 180 cctcccttcc caactctacg ctgcccatgc atggcatctc tctatctact gtctgctgct 240 atactactgt gatgttacta cccttatatg cacagatggg cgttcccgat ctgcgctgtc 300 c 301 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <400> 4 ccacatttcg acctttcaag approx <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <400> 5 ccacatttcg acctttcaag cg <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <400> 6 gaggtcgga gtcaacggat t <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <400> 7 gaaggtgacc aagttcatgc t <210> 8 <211> 43 <212> DNA <213> Artificial Sequence <400> 8 gaaggtcgga gtcaacggat tccacatttc gacctttcaa gca 43 <210> 9 <211> 43 <212> DNA <213> Artificial Sequence <400> 9 gaaggtgacc aagttcatgc tccacatttc gacctttcaa gcg 43 <210> 10 <211> twenty four <212> DNA <213> Artificial Sequence <400> 10 tgctctttga aaagctttgg aaca 24 <210> 11 <211> twenty four <212> DNA <213> Artificial Sequence <400> 11 gaaactgatg tgcgcaaaga gatg 24 <210> 12 <211> twenty four <212> DNA <213> Artificial Sequence <400> 12 gaaactgatg tgcgcaaaga gatt 24 <210> 13 <211> 45 <212> DNA <213> Artificial Sequence <400> 13 gaaggtcgga gtcaacggat tgaaactgat gtgcgcaaag agatg 45 <210> 14 <211> 45 <212> DNA <213> Artificial Sequence <400> 14 gaaggtgacc aagttcatgc tgaaactgat gtgcgcaaag agatt 45 <210> 15 <211> twenty four <212> DNA <213> Artificial Sequence <400> 15 ccagttgttt ctgtagttat ccgc 24 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <400> 16 ccgtcgtcta cagccccc 18 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <400> 17 ccgtcgtcta cagccccg 18 <210> 18 <211> 39 <212> DNA <213> Artificial Sequence <400> 18 gaaggtcgga gtcaacggat tccgtcgtct acagccccc 39 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <400> 19 gaaggtgacc aagttcatgc tccgtcgtct acagccccg 39 <210> 20 <211> twenty four <212> DNA <213> Artificial Sequence <400> 20 gcagacagta gatagagaga tgcc 24

Claims

1. Application of molecular markers Zyf788, Zyf792 and / or Zyf7105, which are closely linked to major QTLs associated with maize ear rot resistance, in any of the following: 1) Application in identifying or assisting in the identification of maize ear rot resistance; 2) Application in maize breeding, wherein the breeding is for cultivating maize with high resistance to ear rot; 3) Application in screening or identifying maize varieties resistant to ear rot; The molecular marker Zyf788 is a SNP site in the maize genome. The molecular marker Zyf788 is located at nucleotide 151 of SEQ ID No. 1 on chromosome 7 of the maize genome, and it is either A or G. The molecular marker Zyf792 is a SNP site in the maize genome. The molecular marker Zyf792 is located at nucleotide 151 of SEQ ID No. 2 on chromosome 7 of the maize genome, and it is either G or T. The molecular marker Zyf7105 is a SNP site in the maize genome. The molecular marker Zyf7105 is located at nucleotide 151 of SEQ ID No. 3 on chromosome 7 of the maize genome, and it is either C or G. The ear rot resistance of maize with the molecular marker Zyf788 genotype GG is lower than that of maize with the molecular marker Zyf788 genotypes AG and AA; the ear rot resistance of maize with the molecular marker Zyf792 genotype TT is lower than that of maize with the molecular marker Zyf792 genotypes GT and GG; the ear rot resistance of maize with the molecular marker Zyf7105 genotype GG is lower than that of maize with the molecular marker Zyf7105 genotypes GC and GG.

2. The application of substances detecting the polymorphism or genotype of the molecular marker Zyf788 as described in claim 1, substances detecting the polymorphism or genotype of the molecular marker Zyf792 as described in claim 1, and / or substances detecting the polymorphism or genotype of the molecular marker Zyf7105 as described in claim 1 in the maize genome in the preparation of products for identifying or assisting in the identification of maize ear rot resistance, characterized in that, The substance used to detect the polymorphism or genotype of the molecular marker Zyf788 in the maize genome is a primer pair for amplifying maize genomic DNA fragments including Zyf788. The two forward-specific fluorescent primer sequences are shown in SEQ ID No. 8 and SEQ ID No. 9, and the reverse universal primer sequence is shown in SEQ ID No.

10. The substance used to detect the polymorphism or genotype of the molecular marker Zyf792 in the maize genome is a primer pair for amplifying maize genomic DNA fragments including Zyf792. The two forward-specific fluorescent primer sequences are shown in SEQ ID No. 13 and SEQ ID No. 14, and the reverse universal primer sequence is shown in SEQ ID No.

15. The substance used to detect the polymorphism or genotype of the molecular marker Zyf7105 in the maize genome is a primer pair for amplifying maize genomic DNA fragments including Zyf7105. The two forward-specific fluorescent primer sequences are shown in SEQ ID No. 18 and SEQ ID No. 19, and the reverse universal primer sequence is shown in SEQ ID No.

20.

3. A method for identifying or assisting in the identification of maize ear rot resistance, characterized in that, The genotypes of the molecular markers Zyf788, Zyf792, and / or Zyf7105 as described in claim 1 were detected in the genome of the maize to be tested. Maize with the genotype GG of molecular marker Zyf788 has lower resistance to ear rot than maize with the genotypes AG and AA of molecular marker Zyf788; maize with the genotype TT of molecular marker Zyf792 has lower resistance to ear rot than maize with the genotypes GT and GG of molecular marker Zyf792; and maize with the genotype GG of molecular marker Zyf7105 has lower resistance to ear rot than maize with the genotypes GC and GG of molecular marker Zyf7105.

4. The application of detecting polymorphisms or genotypes of the molecular marker Zyf788 as described in claim 1, the molecular marker Zyf792 as described in claim 1, and / or the molecular marker Zyf7105 as described in claim 1 in maize genome in maize breeding; wherein the breeding is for cultivating maize with high resistance to ear rot; The substance used to detect the polymorphism or genotype of the molecular marker Zyf788 in the maize genome is a primer pair for amplifying maize genomic DNA fragments including Zyf788. The two forward-specific fluorescent primer sequences are shown in SEQ ID No. 8 and SEQ ID No. 9, and the reverse universal primer sequence is shown in SEQ ID No.

10. The substance used to detect the polymorphism or genotype of the molecular marker Zyf792 in the maize genome is a primer pair for amplifying maize genomic DNA fragments including Zyf792. The two forward-specific fluorescent primer sequences are shown in SEQ ID No. 13 and SEQ ID No. 14, and the reverse universal primer sequence is shown in SEQ ID No.

15. The substance used to detect the polymorphism or genotype of the molecular marker Zyf7105 in the maize genome is a primer pair for amplifying maize genomic DNA fragments including Zyf7105. The two forward-specific fluorescent primer sequences are shown in SEQ ID No. 18 and SEQ ID No. 19, and the reverse universal primer sequence is shown in SEQ ID No.

20. The ear rot resistance of maize with the molecular marker Zyf788 genotype GG is lower than that of maize with the molecular marker Zyf788 genotypes AG and AA; the ear rot resistance of maize with the molecular marker Zyf792 genotype TT is lower than that of maize with the molecular marker Zyf792 genotypes GT and GG; the ear rot resistance of maize with the molecular marker Zyf7105 genotype GG is lower than that of maize with the molecular marker Zyf7105 genotypes GC and GG.

5. A kit for identifying or assisting in the identification of maize ear rot resistance, the kit comprising substances for detecting polymorphisms or genotypes of the molecular marker Zyf788 as described in claim 1 in the maize genome, substances for detecting polymorphisms or genotypes of the molecular marker Zyf792 as described in claim 1 in the maize genome, and / or substances for detecting polymorphisms or genotypes of the molecular marker Zyf7105 as described in claim 1 in the maize genome; The substance used to detect the polymorphism or genotype of the molecular marker Zyf788 in the maize genome is a primer pair for amplifying maize genomic DNA fragments including Zyf788. The two forward-specific fluorescent primer sequences are shown in SEQ ID No. 8 and SEQ ID No. 9, and the reverse universal primer sequence is shown in SEQ ID No.

10. The substance used to detect the polymorphism or genotype of the molecular marker Zyf792 in the maize genome is a primer pair for amplifying maize genomic DNA fragments including Zyf792. The two forward-specific fluorescent primer sequences are shown in SEQ ID No. 13 and SEQ ID No. 14, and the reverse universal primer sequence is shown in SEQ ID No.

15. The substance used to detect the polymorphism or genotype of the molecular marker Zyf7105 in the maize genome is a primer pair for amplifying maize genomic DNA fragments including Zyf7105. The two forward-specific fluorescent primer sequences are shown in SEQ ID No. 18 and SEQ ID No. 19, and the reverse universal primer sequence is shown in SEQ ID No.

20.

6. The use of the kit according to claim 5 in identifying or assisting in the identification of maize ear rot resistance.

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