QTL related to corn resistance to stem rot, SNP molecular markers closely linked thereto and application thereof

Abstract

The present application relates to corn stem rot disease resistance molecular marker and genetic breeding technical field, specifically relates to QTL related to corn stem rot disease resistance, its close linkage SNP molecular marker and application. The present application provides QTL related to corn stem rot disease resistance, which is located in corn 10th chromosome. The present application also provides SNP molecular markers Ks4 and Ks6 closely linked to corn stem rot disease resistance QTL site. By detecting the above-mentioned SNP molecular marker, the genotype of the corn material to be tested can be accurately identified with high throughput, the stem rot disease resistance material can be screened, and the identification and auxiliary screening of corn stem rot disease resistance phenotype can be carried out at seedling stage, which greatly saves the production cost and significantly improves the efficiency of corn breeding resistance to stem rot disease.

Description

Technical Field

[0001] The present invention relates to the technical field of maize molecular markers and genetic breeding, and in particular to QTLs associated with maize stalk rot resistance, tightly linked SNP molecular markers thereof, and applications thereof. Background Art

[0002] corn( Zea mays Maize stalk rot is a major food and feed crop, as well as an important industrial raw material and energy plant. Maize stalk rot is a global soil-borne disease that seriously harms maize production. It causes premature plant aging, stalk lodging, and insufficient grain filling, resulting in yield reduction and restricting mechanized maize harvesting. The dominant pathogen causing maize stalk rot is Fusarium graminearum ( Fusarium graminearum ) and Pythium spp. ( Pythium inflatum Cultivating and promoting varieties resistant to stem rot is the most economical and effective way to prevent and control the disease.

[0003] Because stalk rot is significantly influenced by pathogens, environment, and genotype, and because it is difficult to identify highly resistant or multi-resistant loci, corn stalk rot incidence is increasing year by year. Currently, most identified resistance loci have low efficacy, and relatively few resistance genes have been cloned. Further exploration of maize stalk rot resistance QTLs and resistance genes, and the development of molecular markers tightly linked to QTLs, will provide strong support for molecular breeding for stalk rot resistance by aggregating multiple resistance genes and hopefully improve maize stalk rot resistance.

[0004] Compared with traditional breeding techniques, molecular marker-assisted breeding allows selection to begin at the seedling stage, significantly shortening the breeding cycle. Effective molecular markers are key to molecular marker-assisted breeding. KASP markers do not require typing based on DNA fragment size, eliminating the cumbersome, low-throughput, and expensive nature of traditional gel electrophoresis. They are therefore more suitable for the rapidly developing high-throughput molecular detection platforms. Therefore, developing low-cost, high-throughput molecular detection-compatible KASP markers tightly linked to the QTL for Fusarium graminearum stalk rot resistance in maize is crucial for promoting the application of molecular marker technology and improving the efficiency and level of maize stalk rot resistance breeding in my country. Summary of the Invention

[0005] One of the objects of the present invention is to provide a QTL associated with corn stalk rot resistance and a SNP molecular marker tightly linked thereto. Another object of the present invention is to provide the application of the SNP molecular marker in the identification and breeding of corn stalk rot resistance.

[0006] The development of the maize stalk rot resistance QTL and the molecular markers tightly linked to the QTL provided by the present invention is based on the established recombinant inbred line population, and the stalk rot resistance is identified in multiple environments. Combining high-density genetic mapping and fine mapping, a stable QTL controlling maize stalk rot resistance is located on maize chromosome 10 bin10.06, which is named qGSR10.06 .

[0007] The QTL in the present invention qGSR10.06 The development process of SNP and its linked SNP molecular markers is basically as follows:

[0008] (1) A recombinant inbred line population was constructed to obtain a mapping population: the disease-resistant inbred line KA105, the disease-susceptible inbred line KB204, and 240 F7:8 RIL populations of their combinations.

[0009] (2) The above-mentioned recombinant inbred line population was inoculated with Fusarium graminearum in the field in different years and different environmental locations to identify the occurrence of corn stalk rot in the field, and the stalk rot phenotype was identified according to the stalk rot disease grade. In the present invention, the location and year are uniformly defined as the environment, abbreviated as "year + location initials", such as 2019YL, which means 2019 + Yangling.

[0010] (3) SAS (V9.2) mixed linear models were used to calculate variance components, with genotype as a fixed effect, flowering date as a covariate, and environment, replicates within environment, and genotype-environment interaction as random effects. The broad-sense heritability of stem rot resistance was calculated using the results of the analysis of variance.

[0011] (4) At the 5- to 6-leaf stage, young leaves from multiple plants of consistent growth within each family were mixed and genomic DNA was extracted using the CTAB method. Genotyping analysis was performed using the Maize6H-60K chip independently developed by the Corn Research Center of the Beijing Academy of Agricultural and Forestry Sciences. A high-density linkage map was constructed using QTL ICImapping 4.2 software. Ultimately, a total of 48,087 polymorphic SNP markers were screened and merged into 7,200 bin markers.

[0012] (5) Based on the constructed high-density linkage map, the QTL for resistance to Fusarium graminearum stem rot was detected in combination with the phenotypic values ​​of disease grading in the recombinant inbred line population in single environment and multi-environment combined analysis.

[0013] (6) Select polymorphic SNPs located in the target QTL segment and develop KASP markers for effect verification. Based on the initial QTL mapping results, screen the remaining heterozygous lines in the target segment with fixed background genotypes in the RIL population. Develop KASP markers to screen for individuals that have undergone crossover in the target segment. The key recombinant individuals are verified by the genotype and stem rot phenotype of the offspring for effect verification and fine positioning.

[0014] Using the above technical means, based on the phenotypic data obtained from disease classification in a single environment and a combined analysis of multiple environments, the composite interval mapping method of WinQTLCart V2.5 was used to locate the QTL for resistance to Fusarium graminearum stem rot in maize. In the recombinant inbred line population, the present invention detected a resistance QTL on chromosome 10 bin10.06, named qGSR10.06 The physical position is between 137,771,944 bp–143,942,505 bp (B73 AGP_V3 version), with a confidence interval of 6.17Mb. qGSR10.06 The 2020SY and BLUP values ​​were co-localized, with LOD values ​​ranging from 4.25 to 7.06, explaining 7.78%-8.64% of the phenotypic variation. The resistance allele originated from the disease-resistant parent KA105.

[0015] Further research and development resulted in the identification of two SNP markers closely linked to the QTL locus, namely molecular marker Ks4 and molecular marker Ks6.

[0016] Specifically, the present invention provides the following technical solutions:

[0017] In a first aspect, the present invention provides a QTL associated with corn stalk rot resistance, which is located on corn chromosome 10, with a physical position between 137,771,944 bp and 143,942,505 bp of the B73 AGP_V3 version, and a confidence interval of 6.17 Mb.

[0018] The above QTLs can explain 7.78%-8.64% of the phenotypic variation in maize stalk rot resistance and can be used for positional cloning and molecular marker-assisted selection.

[0019] The SNP molecular markers closely linked to the above-mentioned QTL sites are Ks4 and Ks6. The Ks4 is located at position 142279908 (B73 AGP_V3) of corn chromosome 10, and the mutant base is G or C, that is, the polymorphism is G / C; the Ks6 is located at position 143572421 (B73 AGP_V3) of corn chromosome 10, and the mutant base is A or G, that is, the polymorphism is A / G.

[0020] Furthermore, the present invention provides a QTL associated with corn stalk rot resistance, which is located on chromosome 10 of corn, with a physical position between 142,279,908 bp and 143,572,421 bp of the B73 AGP_V3 version, and a confidence interval of 1.29 Mb.

[0021] In a second aspect, the present invention provides SNP molecular markers tightly linked to a maize stalk rot resistance QTL locus, comprising at least one of the following SNP molecular markers: molecular marker Ks4, molecular marker Ks6;

[0022] The present invention provides SNP molecular markers associated with corn stalk rot resistance, which include at least one of the following SNP molecular markers: molecular marker Ks4, molecular marker Ks6;

[0023] The molecular marker Ks4 is a nucleotide sequence containing a polymorphism of G / C at position 50 of the sequence shown in SEQ ID NO.1; the molecular marker Ks6 is a nucleotide sequence containing a polymorphism of A / G at position 50 of the sequence shown in SEQ ID NO.2.

[0024] Furthermore, the molecular marker Ks4 is a nucleotide sequence containing a polymorphism of G / C at position 60 of the sequence shown in SEQ ID NO.9; and the molecular marker Ks6 is a nucleotide sequence containing a polymorphism of A / G at position 60 of the sequence shown in SEQ ID NO.10.

[0025] The molecular marker Ks4 is a nucleotide sequence containing a polymorphism of G / C at position 70 of the sequence shown in SEQ ID NO.11; the molecular marker Ks6 is a nucleotide sequence containing a polymorphism of A / G at position 70 of the sequence shown in SEQ ID NO.12.

[0026] The molecular marker Ks4 is a nucleotide sequence containing a polymorphism of G / C at position 80 of the sequence shown in SEQ ID NO.13; the molecular marker Ks6 is a nucleotide sequence containing a polymorphism of A / G at position 80 of the sequence shown in SEQ ID NO.14.

[0027] The molecular marker Ks4 is a nucleotide sequence containing a polymorphism of G / C at position 90 of the sequence shown in SEQ ID NO.15; the molecular marker Ks6 is a nucleotide sequence containing a polymorphism of A / G at position 90 of the sequence shown in SEQ ID NO.16.

[0028] The molecular marker Ks4 is a nucleotide sequence containing a polymorphism of G / C at position 100 of the sequence shown in SEQ ID NO.17; the molecular marker Ks6 is a nucleotide sequence containing a polymorphism of A / G at position 100 of the sequence shown in SEQ ID NO.18.

[0029] The molecular marker Ks4 is a nucleotide sequence containing a polymorphism of G / C at position 150 of the sequence shown in SEQ ID NO.19; the molecular marker Ks6 is a nucleotide sequence containing a polymorphism of A / G at position 150 of the sequence shown in SEQ ID NO.20.

[0030] The molecular marker Ks4 is a nucleotide sequence containing a polymorphism of G / C at position 200 of the sequence shown in SEQ ID NO.21; the molecular marker Ks6 is a nucleotide sequence containing a polymorphism of A / G at position 200 of the sequence shown in SEQ ID NO.22.

[0031] The molecular marker Ks4 has the polymorphic site with a genotype of GG, corresponding to a disease-resistant gene; and a genotype of CC, corresponding to a susceptible gene; the disease is corn stalk rot; and the stalk rot is preferably caused by Fusarium graminearum.

[0032] The molecular marker Ks6 has the polymorphic site with a genotype of AA, corresponding to a disease-resistant gene; and a genotype of GG, corresponding to a susceptible gene; the disease is corn stalk rot; and the stalk rot is preferably caused by Fusarium graminearum.

[0033] The above SNP molecular markers and maize stalk rot resistance QTL loci qGSR10.06 Tightly linked.

[0034] Specifically, the molecular marker Ks4 is obtained by PCR amplification using a primer pair as shown in SEQ ID NO.3-5 with corn genomic DNA as a template; further, primer SEQ ID NO.3 and primer SEQ ID NO.4 are respectively labeled with different fluorescent groups;

[0035] The molecular marker Ks6 is obtained by PCR amplification using a primer pair with sequences as shown in SEQ ID NO.6-8 and corn genomic DNA as a template; preferably, primer SEQ ID NO.6 and primer SEQ ID NO.7 are labeled with different fluorescent groups respectively.

[0036] In a preferred embodiment of the present invention, the fluorescent group connected to the 5' end of primer SEQ ID NO.3 and primer SEQ ID NO.6 is FAM, and the sequence is 5'-gaaggtgaccaagttcatgct-3'; the fluorescent group connected to the 5' end of primer SEQ ID NO.4 and primer SEQ ID NO.7 is HEX, and the sequence is 5'-gaaggtcggagtcaacggatt-3'.

[0037] Table 1 Primers for KASP markers used for QTL effect verification

[0038]

[0039] The molecular marker Ks4 has a genotype of GG at the polymorphic site, corresponding to a disease-resistant gene; and a genotype of CC at the polymorphic site, corresponding to a disease-susceptible gene; the disease is corn stalk rot; and the stalk rot is preferably caused by Fusarium graminearum;

[0040] The molecular marker Ks6 has the polymorphic site with a genotype of AA, corresponding to a disease-resistant gene; and a genotype of GG, corresponding to a disease-susceptible gene; the disease is corn stalk rot; and the stalk rot is preferably caused by Fusarium graminearum.

[0041] In a third aspect, the present invention provides primers for amplifying the SNP molecular marker.

[0042] As an embodiment of the present invention, the primers include primers shown as SEQ ID NOs. 3-5, and / or primers shown as SEQ ID NOs. 6-8.

[0043] Furthermore, primer SEQ ID NO.3 and primer SEQ ID NO.4 are respectively labeled with different fluorescent groups; primer SEQ ID NO.6 and primer SEQ ID NO.7 are respectively labeled with different fluorescent groups.

[0044] The present invention also provides a reagent or a kit containing the primer.

[0045] Preferably, the kit may also contain other reagents for PCR amplification, including but not limited to DNA polymerase, PCR reaction buffer, probe, dNTP, Mg 2+ , water, etc.

[0046] In a fourth aspect, the present invention provides any of the following uses of the QTL associated with corn stalk rot resistance, or a molecular marker tightly linked to the QTL, or a SNP molecular marker associated with corn stalk rot resistance, or the primer, or the reagent, or the kit:

[0047] (1) Application in identifying or assisting in identifying resistance to corn stalk rot;

[0048] (2) Application in the preparation of products for identifying or assisting in identifying resistance to corn stalk rot;

[0049] (3) Application in early prediction of corn stalk rot resistance;

[0050] (4) Application in screening corn resistant to stalk rot;

[0051] (5) Application in molecular marker-assisted breeding for corn stalk rot resistance;

[0052] (6) Application in the preparation of products for molecular marker-assisted breeding of corn stalk rot resistance;

[0053] (7) Application in improving maize germplasm resources resistant to stalk rot;

[0054] Among them, the SNP molecular markers are Ks4 and / or Ks6, the Ks4 is located at position 142279908 of corn chromosome 10, the corresponding genome version is B73 AGP_V3, and the polymorphism is G / C; the Ks6 is located at position 143572421 of corn chromosome 10, the corresponding genome version is B73 AGP_V3, and the polymorphism is A / G.

[0055] In a fifth aspect, the present invention provides a method for identifying a corn stalk rot resistance phenotype or screening corn stalk rot resistance, comprising the following steps:

[0056] (1) Extracting genomic DNA from the corn to be identified;

[0057] (2) Using genomic DNA as a template, performing fluorescent quantitative PCR amplification using primers with sequences as shown in SEQ ID NOs. 3-5 and / or primers with sequences as shown in SEQ ID NOs. 6-8;

[0058] (3) Analyze the genotype of the SNP molecular marker in the PCR amplification product, and determine the stalk rot resistance phenotype of the corn to be identified based on the genotype.

[0059] In step (2) of the above method, primer SEQ ID NO.3 and primer SEQ ID NO.4 are respectively labeled with different fluorescent groups; primer SEQ ID NO.6 and primer SEQ ID NO.7 are respectively labeled with different fluorescent groups.

[0060] In step (2), the reaction procedure of the PCR amplification is: pre-denaturation at 94-95°C for 15 min; denaturation at 94-95°C for 20 s, annealing / extension at 61-65°C for 60 s, for a total of 10-13 cycles; denaturation at 94-95°C for 20 s, annealing / extension at 55-57°C for 60 s, for a total of 26-30 cycles.

[0061] In step (3), the genotype of the SNP molecular marker can be analyzed by using a SNP analyzer to detect the fluorescence of the PCR amplification product, and the fluorescence value modified by the primer is read to determine the genotype of the test sample.

[0062] In step (3), the specific method for determining the stalk rot resistance phenotype of the corn to be identified is:

[0063] If the genotype of the polymorphic site of the SNP molecular marker Ks4 is GG, the corn to be identified is corn with stalk rot resistance; if the genotype is CC, the corn to be identified is corn without stalk rot resistance;

[0064] If the genotype of the molecular marker Ks6 at the polymorphic site is AA, the corn to be identified is corn with stalk rot resistance; if the genotype is GG, the corn to be identified is corn without stalk rot resistance.

[0065] In a sixth aspect, the present invention provides a method for breeding corn resistant to stalk rot, the method comprising the steps of causing the corn chromosome to contain the QTL associated with corn stalk rot resistance of the present invention;

[0066] Preferably, the method comprises the step of crossing corn comprising the QTL with corn not comprising the QTL.

[0067] In a seventh aspect, the present invention provides a method for obtaining a corn plant having increased resistance to stalk rot, the method comprising the steps of:

[0068] (1) Providing a first corn plant, which comprises any of the following chromosomal intervals in its genome: a nucleotide sequence corresponding to the 50th base of the sequence shown in SEQ ID NO.1, a nucleotide sequence corresponding to the 60th base of the sequence shown in SEQ ID NO.9, a nucleotide sequence corresponding to the 70th base of the sequence shown in SEQ ID NO.11, a nucleotide sequence corresponding to the 80th base of the sequence shown in SEQ ID NO.13, a nucleotide sequence corresponding to the 90th base of the sequence shown in SEQ ID NO.15, a nucleotide sequence corresponding to the 100th base of the sequence shown in SEQ ID NO.17, a nucleotide sequence corresponding to the 150th base of the sequence shown in SEQ ID NO.19, or a nucleotide sequence corresponding to the 200th base of the sequence shown in SEQ ID NO.21, and / or

[0069] The invention further comprises any of the following chromosomal intervals in its genome: a nucleotide sequence corresponding to the 50th base of the sequence shown in SEQ ID NO. 2, an A-based nucleotide sequence corresponding to the 60th base of the sequence shown in SEQ ID NO. 10, an A-based nucleotide sequence corresponding to the 70th base of the sequence shown in SEQ ID NO. 12, an A-based nucleotide sequence corresponding to the 80th base of the sequence shown in SEQ ID NO. 14, an A-based nucleotide sequence corresponding to the 90th base of the sequence shown in SEQ ID NO. 16, an A-based nucleotide sequence corresponding to the 100th base of the sequence shown in SEQ ID NO. 18, an A-based nucleotide sequence corresponding to the 150th base of the sequence shown in SEQ ID NO. 20, or an A-based nucleotide sequence corresponding to the 200th base of the sequence shown in SEQ ID NO. 22;

[0070] wherein said first corn plant is resistant to stalk rot;

[0071] (2) hybridizing the first corn plant with a second corn plant to obtain a progeny plant; the second corn plant does not contain or contains the chromosome interval described in step (1);

[0072] (3) Selecting progeny plants containing the chromosome interval of step (1) from the progeny plants by isolating nucleic acid from the progeny plants and detecting it in the nucleic acid, thereby obtaining corn plants with increased resistance to stalk rot.

[0073] The corn plants or progeny plants thereof having increased resistance to stalk rot obtained by the above method fall within the scope of protection of the present invention.

[0074] In an eighth aspect, the present invention provides a method for producing a corn plant having stalk rot resistance, the method comprising the steps of:

[0075] (1) Isolation of nucleic acids from corn plants;

[0076] (2) detecting the SNP molecular markers Ks4 and / or Ks6 described in the present invention in the nucleic acid;

[0077] (3) selecting corn plants having stalk rot resistance based on the presence of the SNP molecular marker detected in step (2);

[0078] The selection refers to selecting the corn plant when the genotype of the polymorphic site of the SNP molecular marker Ks4 in the isolated nucleic acid is GG; and / or

[0079] When the genotype of the polymorphic site of the SNP molecular marker Ks6 in the isolated nucleic acid is AA, the corn plant is selected.

[0080] In a ninth aspect, the present invention provides a method for conferring stalk rot resistance to corn, the method comprising: 1) providing a nucleic acid molecule from a chromosome of corn having stalk rot resistance, and 2) inserting the nucleic acid molecule into a chromosome of a recipient corn, thereby producing a corn plant having increased stalk rot resistance compared to the recipient corn;

[0081] The nucleic acid molecule comprises the nucleotide sequence at positions 142279908-143572421 of chromosome 10 of corn or any part thereof, and the nucleic acid molecule can confer resistance to corn stalk rot.

[0082] The corn plants or progeny plants produced by the above method fall within the scope of protection of the present invention.

[0083] In a tenth aspect, the present invention provides a corn plant obtained by hybridizing a first corn plant and a second corn plant, wherein the first corn plant comprises any of the following chromosome intervals in its genome:

[0084] a nucleotide sequence corresponding to the 50th base of the sequence shown in SEQ ID NO.1, a nucleotide sequence corresponding to the 60th base of the sequence shown in SEQ ID NO.9, a nucleotide sequence corresponding to the 70th base of the sequence shown in SEQ ID NO.11, a nucleotide sequence corresponding to the 80th base of the sequence shown in SEQ ID NO.13, a nucleotide sequence corresponding to the 90th base of the sequence shown in SEQ ID NO.15, a nucleotide sequence corresponding to the 100th base of the sequence shown in SEQ ID NO.17, a nucleotide sequence corresponding to the 150th base of the sequence shown in SEQ ID NO.19, or a nucleotide sequence corresponding to the 200th base of the sequence shown in SEQ ID NO.21, and / or

[0085] The invention further comprises any of the following chromosomal intervals in its genome: a nucleotide sequence corresponding to the 50th base of the sequence shown in SEQ ID NO. 2, an A-based nucleotide sequence corresponding to the 60th base of the sequence shown in SEQ ID NO. 10, an A-based nucleotide sequence corresponding to the 70th base of the sequence shown in SEQ ID NO. 12, an A-based nucleotide sequence corresponding to the 80th base of the sequence shown in SEQ ID NO. 14, an A-based nucleotide sequence corresponding to the 90th base of the sequence shown in SEQ ID NO. 16, an A-based nucleotide sequence corresponding to the 100th base of the sequence shown in SEQ ID NO. 18, an A-based nucleotide sequence corresponding to the 150th base of the sequence shown in SEQ ID NO. 20, or an A-based nucleotide sequence corresponding to the 200th base of the sequence shown in SEQ ID NO. 22;

[0086] The first corn plant is resistant to stalk rot;

[0087] The second corn plant contains none or any of the chromosome intervals described above.

[0088] Furthermore, in the corn plant provided by the present invention, the chromosome interval comprises a nucleic acid sequence that is at least 85%, at least 90% or at least 95% identical to the sequence shown in SEQ ID NO.1, and / or comprises a nucleic acid sequence that is at least 85%, at least 90% or at least 95% identical to the sequence shown in SEQ ID NO.2.

[0089] The beneficial effects of the present invention are as follows: the present invention provides a newly discovered maize stalk rot resistance QTL, named qGSR10.06 The LOD values ​​ranged from 4.25 to 7.06, explaining 7.78% to 8.64% of the phenotypic variation. Two SNP molecular markers closely linked to the QTL loci were developed.

[0090] After extensive testing, the present invention found that when using the aforementioned molecular markers to identify or screen for corn stalk rot resistance, the accuracy of identifying stalk rot resistance was approximately 58% when Ks4 was used alone; the accuracy of identifying stalk rot resistance was approximately 50% when Ks6 was used alone; and the accuracy of identifying stalk rot resistance was 84% ​​when both SNP markers were used simultaneously. This indicates that the two SNP molecular markers are effective for assisting selection of corn stalk rot resistance phenotypes, and further demonstrates that both molecular markers Ks4 and Ks6 can be used alone to identify corn stalk rot resistance, and that the combined use of molecular markers Ks4 and Ks6 can significantly improve the accuracy of identifying corn stalk rot resistance.

[0091] The SNP sites in the present invention are clearly located, the detection method is convenient and rapid, is not affected by the environment, has a stronger purpose, a small workload, higher efficiency, and low cost. Therefore, by detecting the SNP sites, identification and auxiliary screening can be carried out at the seedling stage, greatly saving production costs and improving selection efficiency. In corn breeding, the molecular markers and their detection methods of the present invention can be selected to identify corn resistant to stalk rot for breeding, which can improve the selection efficiency of corn breeding and accelerate the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 The disease manifestations of Fusarium graminearum stem rot of the parental inbred lines in Example 1 of the present invention are shown below; wherein A represents the field phenotype of stem rot of the parental inbred lines (HR: disease resistant, HS: disease susceptible); B represents the occurrence of stem rot of the parental inbred lines observed by splitting stems; C represents the disease classification of the parental inbred lines under four environments; and D represents the disease classification standard of stem rot. It indicates significant at the 0.001 level.

[0093] Figure 2 This is a frequency distribution diagram of the disease classification of Fusarium graminearum stem rot of the positioning population in Example 1 of the present invention under the conditions of single environment and multi-environment joint analysis.

[0094] Figure 3 This figure shows the QTL mapping for Fusarium graminearum stem rot disease grade under single and multi-environment combined analysis conditions in Example 3 of the present invention. The figure shows the QTL mapping results for Fusarium graminearum stem rot-related traits on maize chromosome 10 under different environments; the X-axis indicates the genetic map position in cM; and the Y-axis indicates the LOD value. In the QTL additive effect analysis, the lines represent the additive effect values ​​of different QTLs; the X-axis indicates the genetic map position in cM; and the Y-axis indicates the additive effect value, with positive and negative values ​​indicating the direction of the effect.

[0095] Figure 4 In Example 3 of the present invention qGSR10.06 Schematic diagram of effect verification and fine positioning: Figure A is qGSR10.06 The effect of qGSR10.06 Fine-mapping of the resistant allele and the susceptible allele of each recombinant plant. If there is a significant difference in DSI between the two genotypes ( P <0.05), it is inferred that the parental recombinant individuals carry disease-resistant genes, which is represented by R; on the contrary, if there is no significant difference between the two groups of genotypes ( P >0.05), it is inferred that the parental recombinant individuals do not carry the disease resistance gene, represented by S. Black rectangles represent segments homozygous for disease resistance alleles; white rectangles represent segments homozygous for disease susceptible alleles; and gray rectangles represent segments heterozygous for alleles. Molecular markers are used in the analysis. The bar graph shows the DSI distribution of plants with different genotypes. express P <0.05, express P <0.01, express P <0.001. DETAILED DESCRIPTION

[0096] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0097] The KA105 and KB204 used in the following examples are inbred lines independently bred by the maize genetics and breeding team of Northwest Agriculture and Forestry University (Wang Boxin, Wang Yahui, Chen Pengfei, et al. Analysis of combining ability of maize inbred lines derived from Shaanxi A and Shaanxi B groups under different seed density conditions [J]. Acta Agronomica Sinica, 2017, 43(9): 1328-1336). Seeds of the parental inbred lines were provided by the maize breeding team of the College of Agriculture, Northwest Agriculture and Forestry University.

[0098] Fusarium graminearum Fusarium graminearum ) strain was provided by Associate Researcher Long Shusheng from the College of Plant Protection, Northwest Agriculture and Forestry University. This pathogen is the dominant pathogen of stem rot in northern China.

[0099] Example 1 Construction of maize recombinant inbred lines and investigation and phenotypic analysis of their stalk rot resistance traits

[0100] 1. Recombinant inbred line population: 240 F clones of the disease-resistant inbred line KA105 and the susceptible inbred line KB204 and their combinations 7: 8RIL population, see Table 2.

[0101] Table 2 Recombinant inbred line population (240 samples)

[0102]

[0103] 2. Field design

[0104] A population of 240 RILs derived from the parental lines KA105 and KB204, and their combinations, was tested for resistance to stalk rot in Yangling and Sanyuan, Shaanxi Province, from 2019 to 2020. The site and year were collectively defined as an environment, abbreviated as "year + site initials," e.g., 2019YL. Field trials employed an incompletely randomized block design with two replicates per environment. Plants were planted in single-row plots with a row length of 4 m, a row spacing of 0.6 m, and a density of 5,500 plants per mu. Field management practices were the same as those for local field corn production.

[0105] 3. Pathogen culture

[0106] Propagation culture of Fusarium graminearum: The pathogen strain isolated from a single spore is inoculated onto PDA (Potato Dextrose Agar) culture medium and cultured in the dark in a constant temperature incubator at 25°C for 5-7 days. When the mycelium covers the surface of the culture medium, it is cut into equal parts and inoculated onto sterilized corn kernel culture medium together with the culture medium to carry out the propagation of Fusarium graminearum inoculum.

[0107] Preparation of PDA solid medium: Wash, peel, and dice fresh potatoes. Weigh 200 g, place in a pot, add an appropriate amount of pure water, and boil for 30 minutes. Filter through 8 layers of gauze, and collect the filtrate into a 1-liter beaker. Add 20 g of glucose and stir to dissolve. Weigh 15 g of agar powder, heat to dissolve, and pour into the filtrate. Stir thoroughly and adjust the volume to 1 liter. Stir and autoclave at 121°C for 20 minutes. Dispense into Petri dishes in a laminar flow hood and allow to cool and solidify before use. Alternatively, store in a refrigerator at 4°C.

[0108] To prepare the inoculum for Fusarium graminearum: Select healthy, plump corn kernels and soak them in water for 20–22 hours, then boil them in boiling water for 100 minutes. Lay the cooked corn kernels flat and air-dry. Portion the kernels into heat-resistant inoculum bags in 500g increments and autoclave at 121°C for 50 minutes. After cooling, inoculate the inoculum with Fusarium graminearum grown on PDA medium and seal the bags with breathable film. Incubate in the dark at 25°C for 15–20 days until mycelium covers the entire surface of the corn kernels. Rub the bags every two days to ensure adequate contact between the corn kernels and the inoculum. Dry the inoculum in the shade for 3–4 days, then pack them into sacks and store them in a cold storage until ready for use.

[0109] 4. Intra-field bacterial inoculation

[0110] During the tasseling stage, corn is inoculated in the field using the buried root wounding method. Before inoculation, mix the prepared seed inoculation material thoroughly. During inoculation, dig a hole 5–10 cm from the plant's base, cut off some of the fibrous roots to create a wound, place 75–85 infected corn kernels in the hole, and cover with soil, compacting it firmly. After inoculation, irrigate the field to keep the soil moist to promote pathogen growth and infection.

[0111] 5. Investigation of stem rot resistance traits

[0112] 50–55 days after inoculation, corn stalk rot was identified in the field using the stem splitting method. Plants were cut at the cob attachment point and split lengthwise along the stem. The pathogen infection at the root and stem base, as well as the degree of pith necrosis, were observed. The disease rating scale (DRS) was used to identify stalk rot phenotypes, which were divided into five grades:

[0113] Level 1: Highly resistant, normal plants, no obvious symptoms;

[0114] Level 3: disease-resistant, the plant's stem surface is normal, and brown lesions appear at the stem tip;

[0115] Level 5: Moderate resistance, the base of the plant stem becomes soft and faded, with obvious water stains; the pith tissue between the first and second internodes at the base of the stem shrinks and decomposes, and browning occurs;

[0116] Level 7: Susceptible. The lower stem nodes of the plant gradually turn from green to brown. The stem becomes soft and severely dehydrated. The internal stem pith tissue decomposes into filaments and shrinks severely. White or rose-red hyphae appear on the stem nodes. The root system becomes soft, dry, and sparse but does not fall over. The fruit clusters begin to droop.

[0117] Level 9: Highly susceptible, the plant stem is hollow and constricted, with only necrotic vascular tissue remaining inside the stem, the nodes and internodes turn black and have rose-red marks; the root system is sparse, rotten and hollow, the plant is bent or lodged; the bracts of the female ear are dry and loose, and the female ear droops.

[0118] Ten plants of consistent growth were selected from each family. The stem rot disease grade was recorded for each individual plant, and the average value was used as the phenotypic value for each family. The number of days from sowing to 50% of the plants shedding pollen was also investigated and recorded for each family.

[0119] 6. Phenotypic Data Analysis

[0120] In order to evaluate the significance of genotype and environment variance and the interaction between them, the variance components were calculated using the “PROC MIXED” procedure in SAS (V9.2). The mixed linear model was: ,in represents the overall mean of the phenotype, is the genotype effect of the 'i'th family, is the effect of the 'j'th environment, The random residual term is used. Genotype was used as a fixed effect, flowering date as a covariate, and environment, replication within environment, and the interaction between genotype and environment as random effects. Variance components were calculated using a mixed linear model.

[0121] The broad-sense heritability of stem rot resistance was calculated using the variance analysis results. H 2 ): ,in, is the genetic variance, represents the genotype-environment interaction variance, is the error term, n is the number of environments, and r is the number of replicates. To eliminate the effects of environmental variation on phenotypes, a mixed linear model in SAS (V9.2) was used to estimate the best linear unbiased prediction (BLUP) for each family. BLUP values ​​were used as data for multi-environment joint analysis to plot phenotypic distribution histograms and map QTLs across multiple environments.

[0122] Identification of resistance to Fusarium graminearum stem rot in recombinant inbred line populations and parental materials under different environments ( Figure 1 ), the statistical analysis results showed that there were extremely significant differences in disease grading between parental materials in different groups (Table 3).

[0123] Table 3

[0124]

[0125] Note: 2019YL: Yangling in 2019; 2019SY: Sanyuan in 2019; 2020YL: Yangling in 2020; 2020SY: Sanyuan in 2020; BLUP: multi-environment joint analysis.

[0126] In the RIL population, disease grade showed wide and continuous variation across different environments, indicating that stem rot resistance is a quantitative trait controlled by multiple genes. In the recombinant inbred line population, the variation range of disease grade was 1.00-9.00, with a mean of 2.91-5.43 and a coefficient of variation of 33.27%-65.13%. After calculating the BLUP value to eliminate environmental effects, the coefficient of variation of disease grade in the population was reduced to 33.26%. Disease grade in a single environment showed a skewed normal distribution ( Figure 2 ), the BLUP values ​​of the joint analysis of multiple environments showed a normal distribution.

[0127] Table 4 Variance and heritability analysis of stem rot phenotypic indicators in multiple environments

[0128]

[0129] Note: Indicates P<0.001.

[0130] The results of the joint analysis of variance (Table 4) showed that within the mapping population, there were highly significant differences between genotypes, between environments, and in the genotype-by-environment interaction. The estimated genetic variance was greater than the estimated variance for both the environment and the genotype-by-environment interaction, indicating that genetic factors were the primary source of variation, with significant environmental influences. The heritability of stem rot resistance in the recombinant inbred line population was 83.23%.

[0131] Example 2 Maize linkage map construction

[0132] At the 5-6 leaf stage, young leaves from five plants of consistent growth from each family in the RIL population described in Example 1 were mixed and genomic DNA was extracted using the conventional CTAB method. Genotypic data were sequenced by Liaoning East Asia Crop Seed Quality Inspection and Testing Co., Ltd., and genotyping analysis was performed using the Maize6H-60K chip, independently developed by the Corn Research Center of the Beijing Academy of Agricultural and Forestry Sciences. Linkage maps were constructed using QTL ICImapping 4.2 software. The ".map" function was used to construct linkage maps, and the Kosambi function was used to estimate genetic distances. Ultimately, a total of 48,087 polymorphic SNP markers were identified and merged into 7,200 bin markers. A high-density linkage map was constructed using 240 families and 7,200 bin markers. The total map length was 16,501 cM, with an average genetic distance between adjacent markers of 2.29 cM.

[0133] Example 3 Mapping of QTLs for Stalk Rot Resistance in Maize and Mining of Linked SNPs

[0134] 1. QTL positioning

[0135] Based on the linkage map constructed in Example 2, combined with phenotypic values ​​from disease grading in RIL populations analyzed in single and multiple environments, QTLs for Fusarium graminearum stem rot resistance were detected. Initial mapping and genetic effect analysis were performed using the Composite Interval Mapping (CIM) method using Windows QTL Cartographer V2.5 software. The LOD threshold was set at 3, and the confidence interval of the target QTL was determined by decreasing by two LOD values ​​on either side of the LOD peak. QTLs identified simultaneously in multiple environments or those explaining greater than 10% of phenotypic variation in a single environment were selected for effect verification and fine mapping.

[0136] Using the phenotypic data of disease classification in single environment and multi-environment joint analysis, WinQTLCart V 2.5 The QTL mapping for resistance to Fusarium graminearum stem rot in maize was performed using the composite interval mapping method.

[0137] In the mapping population, a resistance QTL was detected on chromosome 10, bin 10.06 ( Figure 3 ), named qGSR10.06 The physical position is between 137,771,944bp–143,942,505 bp (B73 AGP_V3), with a confidence interval of approximately 6.17Mb. qGSR10.06 The 2020SY and BLUP values ​​colocalized (Table 5), with LOD values ​​ranging from 4.25 to 7.06, explaining 7.78%-8.64% of the phenotypic variation, and the resistance allele originated from the disease-resistant parent KA105.

[0138] Table 5 QTL analysis of disease grade of Fusarium graminearum stem rot under single and combined environments

[0139]

[0140] 2. QTL qGSR10.06 Effect verification and fine positioning

[0141] The QTL effect was verified and finely positioned by the recombinant progeny verification method. Based on the initial QTL positioning results, the remaining heterozygous lines with fixed background genotypes in the target segment were screened in the RIL population, and KASP markers were developed to screen for individuals that exchanged in the target segment. The key recombinant individuals were verified by the genotype and stem rot phenotype of the offspring for effect verification and fine positioning. 50-160 plants of each recombinant individual were planted for genotype and stem rot disease grading, and the disease severity index (DSI) of each genotype was calculated. The difference in resistance to Fusarium graminearum stem rot of corn between different genotypes was analyzed using a one-tailed t-test, and the difference between different genotypes was significant ( P <0.05) indicates that the major QTL is located in the heterozygous region, and vice versa.

[0142]

[0143] One recombinant inbred line was screened out from the population qGSR10.06 The same method was used to identify the remaining heterozygous material 638RIL-17-1. qGSR10.06 The results showed that the homozygous allele from the disease-resistant parent can significantly improve the field resistance to stem rot ( P <0.001) ( Figure 4 Figure A). The average DSI of the homozygous disease-resistant KA105 / KA105 genotype plants in Yangling in 2021 was 23.46%, while the average DSI of the homozygous susceptible KB204 / KB204 genotype plants was 75.48%. In 2022, the average DSI of the homozygous KA105 genotype plants in Yangling was 43.29%, while the average DSI of the homozygous KB204 genotype plants was 56.96%. The t-test results showed that there was a very significant difference in DSI between the two genotypes ( P <0.001). In summary, qGSR10.06 Effect detection was carried out, and the results of the two-year experiment showed significant differences, indicating that the genetic effect really exists. The allele derived from KA105 can stably reduce the DSI by 13.67%-52.02%, significantly improving the plant's field resistance to Fusarium graminearum stem rot.

[0144] exist qGSR10.06Molecular markers were encrypted within the candidate interval and molecular marker primers were designed based on the maize B73 reference genome V3 version sequence. The molecular markers at both ends of the candidate interval were Ks4 and Ks6. Ks4 was located at position 142279908 of maize chromosome 10, with a mutation base of G or C; Ks6 was located at position 143572421 of maize chromosome 10, with a mutation base of A or G. The heterozygous offspring produced by 638RIL-17-1 were divided into different recombinant types. qGSR10.06 Fine mapping results showed that there were significant differences in DSI between plants carrying the homozygous KA105 genotype and the homozygous KB204 genotype in types II, III, IV, and V ( P <0.05), the DSI difference between the homozygous genotype plants in types I and VI was not significant ( P >0.05), thus judging that the KA105 donor fragment in the overlapping heterozygous segments of types II, III, IV, and V carries qGSR10.06 ( Figure 4 Figure B). According to whether each type carries qGSR10.06 It can be inferred that this locus is located between markers Ks4 and Ks6 in the heterozygous interval of types I and IV, with a physical distance of approximately 1.29Mb (B73 AGP_V3). Among them, there is a significant difference in DSI between heterozygous genotypes and homozygous susceptible genotypes in type IV ( P <0.05) ( Figure 4 Figure C), in summary, qGSR10.06 It was located between molecular markers Ks4 and Ks6, with a physical distance of about 1.29Mb. The allele from the disease-resistant parent can significantly improve field resistance to stem rot.

[0145] 3. KASP molecular marker development

[0146] Polymorphic SNPs located within the target QTL region were selected to develop KASP markers for effect verification. KASP marker primer design parameters were as follows: primer length 20-27 bp, GC content between 40% and 60%, and annealing temperature between 59°C and 65°C (optimum temperature 63°C). The designed primers are shown in Table 1. For application in KASP technology, a fluorescent linker sequence was added to the 5' end of the designed forward primer: the FAM linker sequence was 5'-gaaggtgaccaagttcatgct-3', and the HEX linker sequence was 5'-gaaggtcggagtcaacggatt-3'. Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. In this example, the primer sequences used to amplify Ks4 are shown in SEQ ID NOs. 23, 24, and 5, respectively; the primer sequences used to amplify Ks6 are shown in SEQ ID NOs. 25, 26, and 8, respectively.

[0147] The PCR reaction system is as follows: a total of 4 μL, including 2 μL HiGeno 2× Probe Mix, 1 μL DNA template (20-50 ng / μL), 0.944 μL ddH₂O, and 0.056 μL primer mix. The preferred primer mix ratio is: 12 μL each of 100 μM forward primers Primer X and Primer Y, 30 μL reverse primer Primer R (100 μM), and 46 μL ddH₂O.

[0148] The PCR reaction program was as follows: pre-denaturation at 94°C for 15 min, first amplification reaction, denaturation at 94°C for 20 s, annealing at 61°C for 60 s, for 10 cycles, and second amplification reaction, denaturation at 94°C for 20 s, annealing and extension at 55°C for 60 s, for 38 cycles.

[0149] PCR products were scanned and fluorescence data were read using a FLUOstar Omega microplate reader (BMG Labtech, Offenburg, Germany). Genotype data were read and clustered using the SNP typing software KclusterCaller (V 3.4.1.36; LGCHoddesdon, UK). The genotype of the FAM allele was X:X, the genotype of the HEX allele was Y:Y, and the heterozygous genotype was X:Y.

[0150] If the genotype of the KASP molecular marker site Ks4 in the maize to be tested is GG, qGSR10.06 The genotype of the tested maize KASP molecular marker site Ks4 is CC, qGSR10.06 The genotype is a susceptible gene.

[0151] If the genotype of the KASP molecular marker site Ks6 in the maize to be tested is AA, qGSR10.06 The genotype of the tested maize KASP molecular marker site Ks6 is GG. qGSR10.06 The genotype is a susceptible gene.

[0152] Example 4 QTL qGSR10.06 Application of tightly linked SNP molecular markers in identification, screening, and breeding of stalk rot-resistant maize varieties

[0153] The material for detecting the polymorphism or genotype of a SNP molecular marker site tightly linked to a QTL for resistance to Fusarium graminearum stalk rot of corn according to the present invention has at least the following applications:

[0154] (1) Identify or assist in identifying corn resistant to stalk rot;

[0155] (2) Preparation of products for identifying or assisting in the identification of corn resistant to stalk rot;

[0156] (3) Corn assisted breeding or preparation of products used for corn assisted breeding.

[0157] The method for identifying or assisting in identifying maize stalk rot (Fusarium graminearum) resistance using molecular markers tightly linked to maize stalk rot resistance QTLs is as follows:

[0158] If the genotype of the molecular marker Ks4 in the maize to be tested is GG, qGSR10.06 The genotype of the molecular marker Ks4 in the maize to be tested is CC. qGSR10.06 The genotype is the susceptible gene;

[0159] If the genotype of the molecular marker Ks6 in the tested corn is AA, qGSR10.06 The genotype of the molecular marker Ks6 in the maize to be tested is GG. qGSR10.06 The genotype is the susceptible gene;

[0160] In summary, corn with the genotype of molecular marker Ks4 being GG and / or the genotype of molecular marker Ks6 being AA was selected as stalk rot-resistant parents for breeding.

[0161] The SNP molecular markers developed in Example 3 were used to screen maize stalk rot-resistant materials. The test materials were the recombinant inbred line population except qGSR10.06Fine-mapping of the remaining 239 recombinant inbred families, excluding family 638RIL-17-1, revealed 74 recombinant inbred lines that were homozygous for the Ks4 and Ks6 molecular markers. Phenotypic identification of stem rot revealed that when the genotypes of the molecular markers Ks4 and Ks6 were both homozygous for the disease-resistant alleles GG and AA, 38 of the 43 recombinant inbred lines showed high resistance to stem rot, with disease grades ranging from 2.42 to 3.92, and an accuracy rate of 88% for stem rot resistance identification.

[0162] When only the molecular marker Ks4 was homozygous for the disease resistance allele GG, 10 of the 14 recombinant inbred lines showed resistance to stem rot, with disease grade ranging from 1.96 to 4.41, and an accuracy rate of 71% for stem rot resistance identification.

[0163] When only the molecular marker Ks6 was homozygous for the disease resistance allele AA, 15 of the 24 recombinant inbred lines showed resistance to stem rot, with disease grade ranging from 2.71 to 4.35, and an accuracy rate of 63% for stem rot resistance identification.

[0164] When the genotypes of molecular markers Ks4 and Ks6 were both homozygous susceptible alleles CC and GG, 28 out of 38 recombinant inbred lines were susceptible to stem rot, with disease grades ranging from 5.18 to 7.86, and the accuracy rate of stem rot susceptibility identification was 74%.

[0165] Table 6 qGSR10.06 Allele type and phenotype at marker

[0166]

[0167] Note: Ks4: GG (resistance allele), CC (susceptibility allele); Ks6: AA (resistance allele), GG (susceptibility allele)

[0168] Table 7 qGSR10.06 Accuracy of identification of resistance to stem rot

[0169]

[0170] Note: In the estimation of the accuracy of stem rot resistance identification, the recombinant inbred lines showing high resistance and resistance are considered to be disease-resistant materials. Ks4: GG (disease resistance allele), CC (disease susceptible allele); Ks6: AA (disease resistance allele), GG (disease susceptible allele)

[0171] qGSR10.06The molecular markers at either end of the candidate interval are Ks4 and Ks6. When Ks4 was used alone, the accuracy of identifying stalk rot resistance was approximately 71%; when Ks6 was used alone, the accuracy was approximately 63%; and when both were used simultaneously, the accuracy of identifying stalk rot resistance was 88%. This demonstrates that the two SNP molecular markers developed in this study are effective for assisting selection of maize stalk rot resistance phenotypes. They can be used alone or in combination for early or assistive identification of maize stalk rot resistance phenotypes, significantly reducing production costs, improving selection efficiency, and accelerating maize breeding.

[0172] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Use of SNP molecular markers associated with corn stalk rot resistance, or primers for amplifying said molecular markers, or reagents or kits containing said primers in identifying or assisting in identifying corn stalk rot resistance; wherein: The SNP molecular marker associated with corn stalk rot resistance includes at least one of the following molecular markers: molecular marker Ks4, molecular marker Ks6; The nucleotide sequence of the molecular marker Ks4 is the sequence shown in SEQ ID NO.1, and the polymorphism at position 50 is G / C; The nucleotide sequence of the molecular marker Ks6 is the sequence shown in SEQ ID NO. 2, and the polymorphism at position 50 is A / G.

2. Use of SNP molecular markers associated with corn stalk rot resistance, or primers for amplifying said molecular markers, or reagents or kits containing said primers in the preparation of products for identifying or assisting in identifying corn stalk rot resistance; wherein: The SNP molecular marker associated with corn stalk rot resistance includes at least one of the following molecular markers: molecular marker Ks4, molecular marker Ks6; The nucleotide sequence of the molecular marker Ks4 is the sequence shown in SEQ ID NO.1, and the polymorphism at position 50 is G / C; The nucleotide sequence of the molecular marker Ks6 is the sequence shown in SEQ ID NO. 2, and the polymorphism at position 50 is A / G.

3. Use of SNP molecular markers associated with corn stalk rot resistance, or primers for amplifying said molecular markers, or reagents or kits containing said primers in the early prediction of corn stalk rot resistance; wherein, The SNP molecular marker associated with corn stalk rot resistance includes at least one of the following molecular markers: molecular marker Ks4, molecular marker Ks6; The nucleotide sequence of the molecular marker Ks4 is the sequence shown in SEQ ID NO.1, and the polymorphism at position 50 is G / C; The nucleotide sequence of the molecular marker Ks6 is the sequence shown in SEQ ID NO. 2, and the polymorphism at position 50 is A / G.

4. Use of SNP molecular markers associated with corn stalk rot resistance, or primers for amplifying said molecular markers, or reagents or kits containing said primers in screening corn for resistance to stalk rot; wherein, The SNP molecular marker associated with corn stalk rot resistance includes at least one of the following molecular markers: molecular marker Ks4, molecular marker Ks6; The nucleotide sequence of the molecular marker Ks4 is the sequence shown in SEQ ID NO.1, and the polymorphism at position 50 is G / C; The nucleotide sequence of the molecular marker Ks6 is the sequence shown in SEQ ID NO. 2, and the polymorphism at position 50 is A / G.

5. Use of SNP molecular markers associated with corn stalk rot resistance, or primers for amplifying said molecular markers, or reagents or kits containing said primers in molecular marker-assisted breeding for corn stalk rot resistance; wherein: The SNP molecular marker associated with corn stalk rot resistance includes at least one of the following molecular markers: molecular marker Ks4, molecular marker Ks6; The nucleotide sequence of the molecular marker Ks4 is the sequence shown in SEQ ID NO.1, and the polymorphism at position 50 is G / C; The nucleotide sequence of the molecular marker Ks6 is the sequence shown in SEQ ID NO. 2, and the polymorphism at position 50 is A / G.

6. Use of SNP molecular markers associated with corn stalk rot resistance, or primers for amplifying said molecular markers, or reagents or kits containing said primers in the preparation of products for molecular marker-assisted breeding of corn stalk rot resistance; wherein: The SNP molecular marker associated with corn stalk rot resistance includes at least one of the following molecular markers: molecular marker Ks4, molecular marker Ks6; The nucleotide sequence of the molecular marker Ks4 is the sequence shown in SEQ ID NO.1, and the polymorphism at position 50 is G / C; The nucleotide sequence of the molecular marker Ks6 is the sequence shown in SEQ ID NO. 2, and the polymorphism at position 50 is A / G.

7. Use of SNP molecular markers associated with corn stalk rot resistance, or primers for amplifying said molecular markers, or reagents or kits containing said primers in improving corn stalk rot resistance germplasm resources; in, The SNP molecular marker associated with corn stalk rot resistance includes at least one of the following molecular markers: molecular marker Ks4, molecular marker Ks6; The nucleotide sequence of the molecular marker Ks4 is the sequence shown in SEQ ID NO.1, and the polymorphism at position 50 is G / C; The nucleotide sequence of the molecular marker Ks6 is the sequence shown in SEQ ID NO. 2, and the polymorphism at position 50 is A / G.

8. A method for identifying corn stalk rot resistance phenotype, characterized in that: The steps include: (1) Extracting genomic DNA from the corn to be identified; (2) Using genomic DNA as a template, performing fluorescent quantitative PCR amplification using primers with sequences as shown in SEQ ID NOs. 3-5 and / or primers with sequences as shown in SEQ ID NOs. 6-8; (3) analyzing the genotype of the SNP molecular marker in the PCR amplification product, and determining the stalk rot resistance phenotype of the corn to be identified based on the genotype; the SNP molecular marker includes at least one of the following molecular markers: molecular marker Ks4 and molecular marker Ks6; The nucleotide sequence of the molecular marker Ks4 is the sequence shown in SEQ ID NO.1, and the polymorphism at position 50 is G / C; The nucleotide sequence of the molecular marker Ks6 is the sequence shown in SEQ ID NO.2, and the polymorphism at position 50 is A / G; In step (3), the method for determining the stalk rot resistance phenotype of the corn to be identified is: If the genotype of the polymorphic site of the molecular marker Ks4 is GG, the corn to be identified is corn with stalk rot resistance; if the genotype is CC, the corn to be identified is corn without stalk rot resistance; If the genotype of the polymorphic site of the molecular marker Ks6 is AA, the corn to be identified is corn with stalk rot resistance; if the genotype is GG, the corn to be identified is corn without stalk rot resistance.

9. The method according to claim 8, characterized in that In step (2), the reaction procedure of the fluorescent quantitative PCR amplification is: pre-denaturation at 94-95°C for 10-15 min; denaturation at 94-95°C for 15-20 s, annealing and extension at 61-65°C for 55-60 s, for a total of 10-13 cycles; denaturation at 94-95°C for 15-20 s, annealing and extension at 55-57°C for 55-60 s, for a total of 38-40 cycles.

10. A method for screening corn plants for resistance to stalk rot, characterized in that The method comprises the following steps: (1) Isolation of nucleic acids from corn plants; (2) detecting a SNP molecular marker in the nucleic acid; the SNP molecular marker comprises at least one of the following molecular markers: molecular marker Ks4 and molecular marker Ks6; the nucleotide sequence of the molecular marker Ks4 is the sequence shown in SEQ ID NO.1, and the polymorphism at position 50 is G / C; the nucleotide sequence of the molecular marker Ks6 is the sequence shown in SEQ ID NO.2, and the polymorphism at position 50 is A / G; (3) selecting corn plants having stalk rot resistance based on the presence of the SNP molecular marker detected in step (2); The selection refers to selecting the corn plant when the genotype of the polymorphic site of the SNP molecular marker Ks4 in the isolated nucleic acid is GG; and / or When the genotype of the polymorphic site of the SNP molecular marker Ks6 in the isolated nucleic acid is AA, the corn plant is selected.

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