QTL related to maize resistance to stalk rot, SNP molecular markers closely linked with QTL, and use of SNP molecular markers

Abstract

The present invention relates to the technical field of molecular markers for maize resistance to stalk rot and genetic breeding, and in particular, to a QTL related to maize resistance to stalk rot, SNP molecular markers closely linked with the QTL, and a use of the SNP molecular markers. Provided is a QTL related to maize resistance to stalk rot, wherein the QTL is located on maize chromosome 10. Also provided are SNP molecular markers Ks4 and Ks6 closely linked with a QTL site for maize resistance to stalk rot. By detecting the SNP molecular markers, the genotype of a maize material under test can be accurately identified in a high-throughput manner, a material resistant to stalk rot is selected, and phenotype identification and assisted screening of the maize resistance to stalk rot can also be carried out in a seedling stage, thus significantly reducing production cost and significantly enhancing the efficiency of breeding maize resistant to stalk rot.

Description

QTLs associated with maize stalk rot resistance, their tightly linked SNP molecular markers, and their applications

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 2023114501103, filed on November 2, 2023, entitled “QTL associated with corn stalk rot resistance, its tightly linked SNP molecular markers and their applications,” all disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0003] 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

[0004] Maize (Zea mays L.) 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 severely impacts maize production. It primarily causes premature plant aging, stalk lodging, and insufficient grain filling, resulting in yield losses and hindering mechanized harvesting. The dominant pathogens causing maize stalk rot are Fusarium graminearum and Pythium inflatum. Breeding and disseminating stalk-rot-resistant varieties is the most cost-effective approach to prevention and control.

[0005] 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.

[0006] 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.

[0007] Summary of the Invention

[0008] 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.

[0009] 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 an established recombinant inbred line population, and stalk rot resistance identification is carried out under multiple environments. Combined with high-density genetic maps and fine positioning, a stable QTL controlling maize stalk rot resistance is located on maize chromosome 10 bin10.06, named qGSR10.06.

[0010] The development process of QTL qGSR10.06 and its linked SNP molecular markers in the present invention is basically as follows:

[0011] (1) Recombinant inbred line populations were constructed to obtain the mapping population: the disease-resistant inbred line KA105, the disease-susceptible inbred line KB204, and 240 F7:8 RIL populations of their combinations.

[0012] (2) The above-mentioned recombinant inbred line populations were 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 to identify the stalk rot phenotype 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.

[0013] (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-by-environment interactions as random effects. The broad-sense heritability of stem rot resistance was calculated using the results of the analysis of variance.

[0014] (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.

[0015] (5) Based on the constructed high-density linkage map, the phenotypic values ​​of disease grading in the recombinant inbred line population in single environment and multi-environment combined analysis were used to detect QTLs for resistance to Fusarium graminearum stem rot.

[0016] (6) Select polymorphic SNPs located in the target QTL segment and develop KASP markers for effect verification. Based on the initial QTL mapping results, select 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.

[0017] Using the above-mentioned technical means, based on the phenotypic data obtained from disease classification in single environments and 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. The present invention detected a resistance QTL on chromosome 10, bin 10.06, in a recombinant inbred line population. The physical position was between 137,771,944bp and 143,942,505bp (B73 AGP_V3 version), with a confidence interval of 6.17Mb. qGSR10.06 co-localized with the 2020SY and BLUP values, with LOD values ​​ranging from 4.25 to 7.06, explaining 7.78% to 8.64% of the phenotypic variation. The resistance allele originated from the disease-resistant parent KA105.

[0018] 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.

[0019] Specifically, the present invention provides the following technical solutions:

[0020] In a first aspect, 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 137,771,944 bp and 143,942,505 bp of the B73 AGP_V3 version, and a confidence interval of 6.17 Mb.

[0021] The above QTL can explain 7.78%-8.64% of the phenotypic variation of maize stalk rot resistance and can be used for map-based cloning and molecular marker-assisted selection.

[0022] 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.

[0023] Furthermore, the present invention provides a QTL associated with corn stalk rot resistance, which is located on corn chromosome 10, 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.

[0024] 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;

[0025] 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;

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] The above-mentioned SNP molecular markers are closely linked to the maize stalk rot resistance QTL locus qGSR10.06.

[0037] 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;

[0038] 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.

[0039] 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'.

[0040] Table 1 Primers for KASP markers used for QTL effect verification

[0041] 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;

[0042] 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.

[0043] In a third aspect, the present invention provides primers for amplifying the SNP molecular marker.

[0044] 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.

[0045] 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.

[0046] The present invention also provides a reagent or a kit containing the primer.

[0047] 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.

[0048] 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:

[0049] (1) Application in identifying or assisting in identifying resistance to corn stalk rot;

[0050] (2) Application in the preparation of products for identifying or assisting in identifying corn stalk rot resistance;

[0051] (3) Application in early prediction of corn stalk rot resistance;

[0052] (4) Application in screening corn for resistance to stalk rot;

[0053] (5) Application of molecular marker-assisted breeding for corn stalk rot resistance;

[0054] (6) Application in the preparation of products for molecular marker-assisted breeding of corn stalk rot resistance;

[0055] (7) Application in improving corn germplasm resources resistant to stalk rot;

[0056] 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.

[0057] 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:

[0058] (1) extracting genomic DNA of the corn to be identified;

[0059] (2) using genomic DNA as a template, performing fluorescent quantitative PCR amplification using primers with sequences shown in SEQ ID NOs. 3-5 and / or primers with sequences shown in SEQ ID NOs. 6-8;

[0060] (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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] In step (3), the specific method for determining the stalk rot resistance phenotype of the corn to be identified is:

[0065] 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;

[0066] 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.

[0067] 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;

[0068] Preferably, the method comprises the step of crossing corn comprising the QTL with corn not comprising the QTL.

[0069] 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:

[0070] (1) Providing a first corn plant, comprising 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

[0071] The present invention 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, a nucleotide sequence corresponding to the 60th base of the sequence shown in SEQ ID NO.10, a nucleotide sequence corresponding to the 70th base of the sequence shown in SEQ ID NO.12, a nucleotide sequence corresponding to the 80th base of the sequence shown in SEQ ID NO.14, a nucleotide sequence corresponding to the 90th base of the sequence shown in SEQ ID NO.16, a nucleotide sequence corresponding to the 100th base of the sequence shown in SEQ ID NO.18, a nucleotide sequence corresponding to the 150th base of the sequence shown in SEQ ID NO.20, or a nucleotide sequence corresponding to the 200th base of the sequence shown in SEQ ID NO.22;

[0072] wherein said first corn plant is resistant to stalk rot;

[0073] (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);

[0074] (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.

[0075] 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.

[0076] 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:

[0077] (1) Isolating nucleic acids from corn plants;

[0078] (2) detecting the SNP molecular markers Ks4 and / or Ks6 of the present invention in the nucleic acid;

[0079] (3) selecting corn plants having stalk rot resistance based on the presence of the SNP molecular marker detected in step (2);

[0080] 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

[0081] 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.

[0082] 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;

[0083] 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.

[0084] The corn plants or progeny plants produced by the above method fall within the scope of protection of the present invention.

[0085] 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:

[0086] 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

[0087] 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;

[0088] The first corn plant is resistant to stalk rot;

[0089] The second corn plant contains none or any of the chromosome intervals described above.

[0090] 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.

[0091] 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, with an LOD value between 4.25-7.06, an explainable phenotypic variation of 7.78%-8.64%, and develops two SNP molecular markers tightly linked to the QTL site.

[0092] After extensive testing, the present invention found that when using the above 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 molecular markers were used for testing. This indicates that the two SNP molecular markers are effective for assisting selection of corn stalk rot resistance phenotypes, and further demonstrates that the use of molecular markers Ks4 and Ks6 alone can 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.

[0093] 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

[0094] Figure 1 shows the symptoms of Fusarium graminearum stem rot of the parental inbred lines in Example 1 of the present invention; wherein, A is the field phenotype of stem rot of the parental inbred lines (HR: disease-resistant, HS: susceptible); B is the observation of the occurrence of stem rot by splitting the stems of the parental inbred lines; C is the disease classification of the parental inbred lines under four environments; D is the disease classification standard for stem rot; *** indicates significance at the 0.001 level.

[0095] FIG2 is a frequency distribution diagram of the disease classification of Fusarium graminearum stem rot of the positioning population under the conditions of single environment and multi-environment joint analysis in Example 1 of the present invention.

[0096] Figure 3 shows the QTL mapping for Fusarium graminearum stem rot disease classification under single and multi-environment combined analysis conditions in Example 3 of the present invention. In the figure, the lines represent the QTL mapping results for Fusarium graminearum stem rot-related traits on maize chromosome 10 under different environments; the X-axis represents the genetic map position in cM; and the Y-axis represents the LOD value. In the additive effect analysis of QTLs, the lines represent the additive effect values ​​expressed by different QTLs; the X-axis represents the genetic map position in cM; and the Y-axis represents the additive effect value, with positive and negative values ​​indicating the direction of the effect.

[0097] Figure 4 is a schematic diagram of the effect verification and fine mapping of qGSR10.06 in Example 3 of the present invention: Panel A shows the effect verification of qGSR10.06; Panel B shows the fine mapping of qGSR10.06; and Panel C shows the distribution of the DSI of the segregating progeny of heterozygous individual plants during fine mapping. The QTL effect verification and fine mapping were performed using the recombinant progeny verification method. A t-test was used to analyze whether there was a difference in the DSI between the progeny homozygous for the disease-resistant allele and the progeny homozygous for the susceptible allele of each recombinant individual. If there was a significant difference in the DSI between the two genotype groups (P < 0.05), the parental recombinant individual was inferred to carry the disease-resistant gene, indicated by R. Conversely, if there was no significant difference between the two genotype groups (P > 0.05), the parental recombinant individual was inferred to not carry the disease-resistant gene, indicated by S. Black rectangles represent segments homozygous for the disease-resistant allele; white rectangles represent segments homozygous for the disease-susceptible allele; and gray rectangles represent segments heterozygous for the heterozygous allele. Molecular markers are the markers used in the analysis. The bar graph shows the DSI distribution of plants with different genotypes. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0098] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0099] 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 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.

[0100] The 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.

[0101] Example 1 Construction of maize recombinant inbred lines and investigation and phenotypic analysis of stalk rot resistance traits

[0102] 1. Recombinant inbred line population: 240 F clones of the disease-resistant inbred line KA105 and the susceptible inbred line KB204 and their combinations 7:8 RIL population, see Table 2.

[0103] Table 2 Recombinant inbred line population (240 copies)

[0104] 2. Field design

[0105] 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.

[0106] 3. Pathogen culture

[0107] 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 to carry out the propagation of Fusarium graminearum inoculum.

[0108] Preparation of PDA solid culture medium: Select fresh potatoes, wash, peel and cut into pieces. Weigh 200 g, put them into a pot, add appropriate amount of pure water and boil for 30 minutes, filter through 8 layers of gauze, and collect the filtrate into a 1L beaker; add 20 g of glucose and stir to dissolve, weigh 15 g of agar powder, heat and dissolve, then pour it into the filtrate, stir evenly and adjust the volume to 1L, sterilize at 121℃ and high temperature and high pressure for 20 minutes, divide into culture dishes on a clean bench, cool and solidify for use, or store in a 4℃ refrigerator for later use.

[0109] 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 full 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.

[0110] 4. Intra-field bacterial inoculation

[0111] 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.

[0112] 5. Investigation of stem rot resistance traits

[0113] 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 longitudinally 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 are divided into five grades:

[0114] Level 1: Highly resistant, normal plants, no obvious symptoms;

[0115] Level 3: disease-resistant, the plant's stem surface is normal, and brown lesions appear at the stem tip;

[0116] 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;

[0117] 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.

[0118] 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.

[0119] Ten plants of consistent growth were selected from each family, and their stem rot disease grade was recorded. 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.

[0120] 6. Phenotypic Data Analysis

[0121] To assess the significance of genotype and environment variance, as well as their interaction, we used SAS (V9.2) using the "PROC MIXED" procedure to calculate variance components. A mixed linear model was constructed as follows: y = μ + gi + ej + ε, where μ represents the grand mean of the phenotype, gi is the genotype effect for the i-th family, ej is the effect for the j-th environment, and ε is a random residual. Variance components were calculated using a mixed linear model with genotype as a fixed effect, flowering date as a covariate, and environment, replicates within environments, and genotype-by-environment interactions as random effects.

[0122] The broad-sense heritability (H) of stem rot resistance was calculated using the variance analysis results. 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 was used in SAS (V9.2) to estimate the best linear unbiased prediction (BLUP) for each family. The BLUP values ​​were used as data for multi-environment joint analysis to plot phenotypic distribution histograms and QTL mapping.

[0123] Resistance to Fusarium graminearum stem rot in recombinant inbred line populations and parental materials was identified under different environments (Figure 1). Statistical analysis results showed that there were extremely significant differences in disease grading between parental materials in different populations (Table 3).

[0124] Table 3

[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, disease grade varied from 1.00 to 9.00, with means ranging from 2.91 to 5.43 and coefficients of variation ranging from 33.27% to 65.13%. After calculating the BLUP value to eliminate environmental influences, the coefficient of variation of disease grade in the population decreased to 33.26%. Disease grade within a single environment exhibited a skewed normal distribution (Figure 2), while the BLUP values ​​for multiple environments showed a normal distribution.

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

[0128] Note: *** indicates P < 0.001.

[0129] 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 stalk rot resistance in the recombinant inbred line population was 83.23%.

[0130] Example 2 Maize linkage map construction

[0131] 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 obtained by sequencing at 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.

[0132] Example 3: Mapping QTLs for resistance to stalk rot in maize and mining linked SNP loci

[0133] 1. QTL positioning

[0134] Based on the linkage map constructed in Example 2, the phenotypic values ​​of disease grading in the RIL population in single and multiple environments were combined to detect QTLs for resistance to Fusarium graminearum stem rot. The composite interval mapping (CIM) method of Windows QTL Cartographer V2.5 software was used for preliminary positioning and genetic effect analysis. The LOD threshold was set to 3, and the confidence interval of the target QTL was determined by dropping 2 LOD values ​​on both sides of the LOD value peak. QTLs identified simultaneously in multiple environments or QTLs with an explanation rate of phenotypic variation greater than 10% in a single environment were selected for effect verification and fine positioning.

[0135] 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.

[0136] In the mapping population, a resistance QTL was detected on chromosome 10, bin 10.06 (Figure 3), designated qGSR10.06, with a physical location between 137,771,944 bp and 143,942,505 bp (B73 AGP_V3), and a confidence interval of approximately 6.17 Mb. qGSR10.06 colocalized with the 2020SY and BLUP values ​​(Table 5), with LOD values ​​ranging from 4.25 to 7.06, explaining 7.78% to 8.64% of the phenotypic variation. The resistance allele originated from the resistant parent, KA105.

[0137] Table 5 QTL analysis of disease grade of Fusarium graminearum stem rot under single and combined environments

[0138] 2. Verification and fine mapping of the QTL qGSR10.06 effect

[0139] The QTL effect verification and fine positioning were performed by the recombinant progeny verification method. According to the results of the initial QTL positioning, the remaining heterozygous lines with fixed background genotypes in the target segment were screened in the RIL population, and the 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 identification, 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. If the difference between different genotypes was significant (P < 0.05), it means that the main effect QTL is located in the heterozygous region, and vice versa. DSI (%) = Σ (disease grade × number of plants with corresponding grade) × 100 / (maximum disease grade × total number of plants with identified stem rot phenotypes)

[0140] A single remaining heterozygous line, 638RIL-17-1, was identified from the recombinant inbred line population, heterozygous for the qGSR10.06 region. The same method was used to validate the effect of qGSR10.06. The results showed that the homozygous allele from the resistant parent significantly improved field resistance to stem rot (P < 0.001) (Figure 4, Panel A). In Yangling in 2021, the mean DSI of the homozygous resistant KA105 / KA105 genotype was 23.46%, while that of the homozygous susceptible KB204 / KB204 genotype was 75.48%. In Yangling in 2022, the mean DSI of the homozygous KA105 genotype was 43.29%, while that of the homozygous KB204 genotype was 56.96%. A t-test revealed a highly significant difference in the DSI between the two genotypes (P < 0.001). In summary, the effect of qGSR10.06 was detected, and the results of the two-year experiment showed significant differences, indicating that the genetic effect is real. 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.

[0141] Molecular markers were encrypted within the candidate interval of qGSR10.06, and primers for molecular markers were designed using the maize B73 reference genome version 3 sequence as a reference. The molecular markers Ks4 and Ks6, located at either end of the candidate interval, were located at position 142279908 on maize chromosome 10, with a mutation of either G or C; and Ks6, located at position 143572421 on maize chromosome 10, with a mutation of either A or G. Heterozygous progeny of 638RIL-17-1 were classified into different recombinant types. Fine-mapping of qGSR10.06 revealed significant differences in the DSI between plants carrying the homozygous KA105 genotype and the homozygous KB204 genotype in groups II, III, IV, and V (P < 0.05). However, no significant differences were observed between plants carrying the homozygous genotypes in groups I and VI (P > 0.05). This suggests that the KA105 donor fragment in the overlapping heterozygous region of groups II, III, IV, and V carries qGSR10.06 (Figure 4B). Based on the presence of qGSR10.06 in each group, it was inferred that the locus is located between markers Ks4 and Ks6 in the heterozygous region of groups I and IV, with a physical distance of approximately 1.29 Mb (B73 AGP_V3). Among them, there was a significant difference in DSI between heterozygous genotype and homozygous susceptible genotype plants in type IV (P < 0.05) (Figure 4, Panel C). In summary, qGSR10.06 was located between molecular markers Ks4 and Ks6, with a physical distance of approximately 1.29 Mb. The allele from the disease-resistant parent can significantly improve field resistance to stem rot.

[0142] 3. KASP molecular marker development

[0143] Polymorphic SNPs located within the target QTL region were selected to develop KASP markers for effect verification. KASP marker primer design parameters are 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 further added to the 5' end of the designed forward primer: the FAM linker sequence is 5'-gaaggtgaccaagttcatgct-3', and the HEX linker sequence is 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.

[0144] 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.

[0145] 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.

[0146] PCR products were scanned and fluorescence data were read using a FLUOstar Omega microplate reader (BMG Labtech, Offenburg, Germany). Genotyping data were read and clustered using the SNP typing software KclusterCaller (V 3.4.1.36; LGC Hoddesdon, 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.

[0147] If the genotype at the KASP molecular marker site Ks4 of the tested corn is GG, the genotype of qGSR10.06 is a disease-resistant gene; if the genotype at the KASP molecular marker site Ks4 of the tested corn is CC, the genotype of qGSR10.06 is a disease-susceptible gene.

[0148] If the genotype at the KASP molecular marker site Ks6 of the tested corn is AA, the genotype of qGSR10.06 is a disease-resistant gene; if the genotype at the KASP molecular marker site Ks6 of the tested corn is GG, the genotype of qGSR10.06 is a disease-susceptible gene.

[0149] Example 4 Application of QTL qGSR10.06 and Tightly Linked SNP Molecular Markers in Identification, Screening, and Breeding of Stalk Rot-Resistant Maize

[0150] 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:

[0151] (1) Identify or assist in identifying corn resistant to stalk rot;

[0152] (2) preparing products for identifying or assisting in identifying corn resistant to stalk rot;

[0153] (3) Corn assisted breeding or preparation of products for corn assisted breeding.

[0154] 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:

[0155] If the genotype of the molecular marker Ks4 in the tested maize is GG, the genotype of qGSR10.06 is a disease-resistant gene; if the genotype of the molecular marker Ks4 in the tested maize is CC, the genotype of qGSR10.06 is a disease-susceptible gene;

[0156] If the genotype of the molecular marker Ks6 in the tested maize is AA, the genotype of qGSR10.06 is a disease-resistant gene; if the genotype of the molecular marker Ks6 in the tested maize is GG, the genotype of qGSR10.06 is a disease-susceptible gene;

[0157] 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.

[0158] The SNP molecular markers developed in Example 3 were used to screen for maize stalk rot-resistant materials. The test materials were 239 recombinant inbred lines from the recombinant inbred line population, excluding the qGSR10.06 fine-mapping family 638RIL-17-1. 74 recombinant inbred lines were screened for homozygous genes at the Ks4 and Ks6 molecular markers. Phenotypic identification of stalk rot revealed that when the genotypes of the molecular markers Ks4 and Ks6 were both homozygous disease-resistant alleles GG and AA, 38 of the 43 recombinant inbred lines showed high resistance to stalk rot, with disease grades ranging from 2.42 to 3.92, and an accuracy rate of stalk rot resistance identification of 88%.

[0159] 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.

[0160] 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.

[0161] 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 grade ranging from 5.18 to 7.86, and the accuracy rate of stem rot susceptible identification was 74%.

[0162] Table 6 Alleles and phenotypes at marker qGSR10.06

[0163] Note: Ks4: GG (resistance allele), CC (susceptibility allele); Ks6: AA (resistance allele), GG (susceptibility allele)

[0164] Table 7 Accuracy of identification of stem rot resistance using the qGSR10.06 marker

[0165] 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)

[0166] The molecular markers Ks4 and Ks6, located at either end of the qGSR10.06 candidate interval, identified stalk rot resistance with an accuracy rate of approximately 71% when Ks4 was used alone; approximately 63% when Ks6 was used alone; and 88% when both markers were used simultaneously. 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 individually 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.

[0167] 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. Industrial Applicability

[0168] The present invention provides a QTL associated with corn stalk rot resistance, a tightly linked SNP molecular marker thereof, and an application thereof. The present invention provides a QTL associated with corn stalk rot resistance, which is located on corn chromosome 10. The present invention also provides SNP molecular markers Ks4 and Ks6 tightly linked to the corn stalk rot resistance QTL locus. By detecting the above-mentioned SNP molecular markers, the genotype of the corn material to be tested can be accurately identified with high throughput, and stalk rot-resistant materials can be screened. The identification and auxiliary screening of the corn stalk rot resistance phenotype can also be carried out at the seedling stage, which greatly saves production costs, significantly improves the efficiency of stalk rot-resistant corn breeding, and has good economic value and application prospects.

Claims

1. A QTL associated with maize resistance to stalk rot, characterized in that: It is located on chromosome 10 of maize, with a physical position between 142,279,908bp–143,572,421bp of the B73 AGP_V3 version, and a confidence interval of 1.29Mb.

2. A SNP molecular marker associated with corn stalk rot resistance, characterized in that: It includes at least one of the following SNP molecular markers: molecular marker Ks4, molecular marker Ks6; 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 the polymorphism A / G at position 50 of the sequence shown in SEQ ID NO.

2.

3. The SNP molecular marker according to claim 2, characterized in that: The molecular marker Ks4 is obtained by PCR amplification using a primer pair with a sequence as shown in SEQ ID NO.3-5 and corn genomic DNA as a template; or 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.

4. The SNP molecular marker according to claim 2 or 3, characterized in that: The molecular marker Ks4 has a genotype of GG at the polymorphic site, corresponding to a disease-resistant gene; and a genotype of CC, corresponding to a disease-susceptible gene; The disease is corn stalk rot; The molecular marker Ks6 has a 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.

5. Primers for amplifying the SNP molecular marker according to any one of claims 2 to 4.

6. The primer according to claim 5, characterized in that The primers include primers with sequences as shown in SEQ ID NO.3-5, and / or primers with sequences as shown in SEQ ID NO.6-8.

7. A reagent or kit comprising the primer according to claim 5 or 6.

8. Any of the following uses of the QTL associated with corn stalk rot resistance according to claim 1 or the SNP molecular marker closely linked to the QTL, or the SNP molecular marker according to any one of claims 2 to 4, or the primer according to claim 5 or 6, or the reagent or kit according to claim 7: (1) Application in identifying or assisting in identifying resistance to corn stalk rot; (2) Application in the preparation of products for identifying or assisting in identifying resistance to corn stalk rot; (3) Application in early prediction of corn stalk rot resistance; (4) Application in screening corn resistant to stalk rot; (5) Application of molecular marker-assisted breeding for maize resistance to stalk rot; (6) Application in the preparation of products for molecular marker-assisted breeding of corn stalk rot resistance; (7) Application in improving maize germplasm resources resistant to stalk rot.

9. A method for identifying the phenotype of corn resistance to stalk rot or screening corn resistance to stalk rot, characterized in that: The steps include: (1) extracting genomic DNA of corn to be identified; (2) using genomic DNA as a template, and 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 according to any one of claims 2 to 4 in the PCR amplification product, and determining the stalk rot resistance phenotype of the corn to be identified based on the genotype; Preferably, 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 38-40 cycles.

10. The method according to claim 9, characterized in that 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 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; 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.

11. A method for breeding corn resistant to stalk rot, characterized in that: The method comprises the steps of making the corn chromosome contain the QTL associated with corn stalk rot resistance according to claim 1; Preferably, the method comprises the step of crossing corn comprising the QTL with corn not comprising the QTL.

12. A method for obtaining a corn plant having increased resistance to stalk rot, characterized in that The method comprises the following steps: (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, a nucleotide sequence corresponding to the 200th base of the sequence shown in SEQ ID NO.21, and / or The invention 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, a nucleotide sequence corresponding to the 60th base of the sequence shown in SEQ ID NO.10, a nucleotide sequence corresponding to the 70th base of the sequence shown in SEQ ID NO.12, a nucleotide sequence corresponding to the 80th base of the sequence shown in SEQ ID NO.14, a nucleotide sequence corresponding to the 90th base of the sequence shown in SEQ ID NO.16, a nucleotide sequence corresponding to the 100th base of the sequence shown in SEQ ID NO.18, a nucleotide sequence corresponding to the 150th base of the sequence shown in SEQ ID NO.20, or a nucleotide sequence corresponding to the 200th base of the sequence shown in SEQ ID NO.22; wherein said first corn plant is resistant to stalk rot; (2) hybridizing the first corn plant with the second corn plant to obtain a progeny plant; the second corn plant does not contain or contains the chromosome interval described in step (1); (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.

13. A method for producing corn plants resistant to stalk rot, characterized in that The method comprises the following steps: (1) Isolating nucleic acids from corn plants; (2) detecting the SNP molecular marker according to claim 2 or 3 in the nucleic acid; (3) selecting corn plants having stalk rot resistance based on the presence of the SNP molecular marker detected in step (2); The selection means selecting the corn plant when the genotype of the site with the polymorphism in the SNP molecular marker Ks4 in the isolated nucleic acid is GG; and / or When the genotype of the site where the SNP molecular marker Ks6 in the isolated nucleic acid has the polymorphism is AA, the corn plant is selected.

14. A method for imparting resistance to corn stalk rot, characterized in that: The method comprises: 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; The nucleic acid molecule comprises the nucleotide sequence at position 142279908-143572421 of chromosome 10 of corn or any part thereof, and the nucleic acid molecule can confer resistance to corn stalk rot.

15. A corn plant obtained by the method of any one of claims 11-14.

16. A corn plant, characterized in that The plant is 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: A nucleotide sequence corresponding to the 50th base of the sequence shown in SEQ ID NO.1, wherein the base is T, or a nucleotide sequence corresponding to the 60th base of the sequence shown in SEQ ID NO.9, wherein the base is G, or a nucleotide sequence corresponding to the 70th base of the sequence shown in SEQ ID NO.11, wherein the base is G, or a nucleotide sequence corresponding to the 80th base of the sequence shown in SEQ ID NO.13, wherein the base is G, or a nucleotide sequence corresponding to the 90th base of the sequence shown in SEQ ID NO.15, wherein the base is G, or a nucleotide sequence corresponding to the 100th base of the sequence shown in SEQ ID NO.17, wherein the base is G, or a nucleotide sequence corresponding to the 150th base of the sequence shown in SEQ ID NO.19, wherein the base is G, or a nucleotide sequence corresponding to the 200th base of the sequence shown in SEQ ID NO.21, and / or The invention 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, a nucleotide sequence corresponding to the 60th base of the sequence shown in SEQ ID NO.10, a nucleotide sequence corresponding to the 70th base of the sequence shown in SEQ ID NO.12, a nucleotide sequence corresponding to the 80th base of the sequence shown in SEQ ID NO.14, a nucleotide sequence corresponding to the 90th base of the sequence shown in SEQ ID NO.16, a nucleotide sequence corresponding to the 100th base of the sequence shown in SEQ ID NO.18, a nucleotide sequence corresponding to the 150th base of the sequence shown in SEQ ID NO.20, or a nucleotide sequence corresponding to the 200th base of the sequence shown in SEQ ID NO.22; The first corn plant is resistant to stalk rot; The second corn plant contains none or any of the chromosome intervals described above.

17. The corn plant of claim 16, wherein: The chromosome interval contains 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 contains 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.

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