QTL (quantitative trait loci) related to corn stem rot resistance, SNP (single nucleotide polymorphism) molecular marker closely linked with QTL and application of QTL

Abstract

The invention relates to the technical field of corn stem rot resistance molecular markers and genetic breeding, in particular to a QTL (Quantitative Trait Loci) related to corn stem rot resistance, an SNP (Single Nucleotide Polymorphism) molecular marker closely linked with the QTL and application of the QTL. The invention provides a QTL (Quantitative Trait Loci) related to corn stem rot resistance. The QTL is located in a corn chromosome 10. The invention also provides SNP (Single Nucleotide Polymorphism) molecular markers Ks4 and Ks6 which are closely linked with the corn stem rot resistant QTL locus. By detecting the SNP molecular marker, the genotype of a to-be-detected corn material can be accurately identified in a high-throughput manner, the stem rot resistant material can be screened, identification and auxiliary screening of the corn stem rot resistant phenotype can also be carried out in the seedling stage, the production cost is greatly saved, and the breeding efficiency of the stem rot resistant corn is remarkably improved.

Description

Technical Field

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

[0002] Corn (Zea mays L.) is a major food and feed crop, as well as an important industrial raw material and energy plant. Corn stalk rot is a global soil-borne disease that seriously harms corn production. It mainly causes premature aging of plants, stalk lodging, and insufficient grain filling, resulting in reduced yields and restricting mechanized corn harvesting. The dominant pathogens that cause corn stalk rot are mainly Fusarium graminearum and Pythium inflatum. Cultivating and promoting varieties resistant to stalk rot is the most economical and effective way to prevent and control it.

[0003] Since stalk rot is greatly affected by pathogens, environment, and genotype, and it is difficult to find highly resistant and multi-resistant sources, the incidence of corn stalk rot has become more serious year by year. At present, most of the resistance loci that have been located have low effects, and few disease resistance genes have been cloned. Further exploration of corn stalk rot resistance QTLs and disease resistance genes, and development of molecular markers closely linked to QTLs, will provide strong support for molecular breeding of stalk rot resistance to aggregate multiple resistance genes, and is expected to improve corn stalk rot resistance.

[0004] Compared with traditional breeding techniques, molecular marker-assisted breeding can start from the seedling stage, greatly shortening the breeding cycle. Effective molecular markers are the key to molecular marker-assisted breeding. KASP markers do not need to be typed according to the size of DNA fragments, and can get rid of the relatively cumbersome, low-throughput, and expensive detection method of traditional gel electrophoresis, which is more suitable for the rapidly developing high-throughput molecular detection platform at this stage. Therefore, the development of low-cost, high-throughput molecular detection platform-suitable KASP molecular markers tightly linked to the maize resistance to Fusarium graminearum stalk rot QTL is of great significance for promoting the application of molecular marker technology and improving the efficiency and level of maize breeding for resistance to stalk rot in my country. Summary of the invention

[0005] One of the purposes of the present invention is to provide a QTL associated with corn stalk rot resistance and a SNP molecular marker closely linked thereto, and another purpose 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 an established recombinant inbred line population, and stalk rot resistance is identified 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.

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

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

[0009] (2) The above-mentioned recombinant inbred line population was inoculated with Fusarium graminearum in different years and different environmental locations, the occurrence of corn stalk rot in the field was identified, and the stalk rot phenotype was identified according to the stalk rot disease classification. The present invention uniformly defines the location and year as the environment, abbreviated as "year + location first letter", such as 2019YL, meaning 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, replication within environment, and the interaction between genotype and environment as random effects. The results of variance analysis were used to calculate the broad-sense heritability of stem rot resistance.

[0011] (4) At the 5-6 leaf stage of corn, young leaves of multiple plants with consistent growth in each family were selected and mixed, and genomic DNA was extracted by CTAB method. Genotyping analysis was performed using Maize6H-60K chip independently developed by the Corn Research Center of Beijing Academy of Agricultural and Forestry Sciences. A high-density linkage map was constructed using QTL ICImapping 4.2 software. Finally, 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, QTL detection for Fusarium graminearum stem rot resistance was carried out in combination with the phenotypic values ​​of disease grading in a recombinant inbred line population in a single environment and in a combined analysis of multiple environments.

[0013] (6) Select polymorphic SNPs located in the target QTL segment and develop KASP markers for effect verification. Based on the initial QTL positioning results, select the remaining heterozygous lines with fixed background genotypes in the target segment in the RIL population, develop KASP markers to screen and obtain individuals that have exchanged in the target segment, and verify the effect and fine-tune the positioning of key recombinant individuals through the genotype and stem rot phenotype of the offspring.

[0014] Using the above technical means, based on the phenotypic data of disease classification in single environment and multi-environment joint analysis, the maize Fusarium graminearum stem rot resistance QTL positioning was carried out by the composite interval mapping method of WinQTLCart V2.5. In the recombinant inbred line population, the present invention detected a resistance QTL on chromosome 10 bin10.06, named qGSR10.06, with a physical position between 137,771,944bp-143,942,505bp (B73 AGP_V3 version), and a confidence interval of 6.17Mb. qGSR10.06 co-localized with 2020SY and BLUP values, with LOD values ​​between 4.25-7.06, and explained phenotypic variation of 7.78%-8.64%, and 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 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.

[0018] The above QTL can explain 7.78%-8.64% of the phenotypic variation of corn 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 loci 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] Further, 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 a SNP molecular marker tightly linked to a maize stalk rot resistance QTL locus, which includes 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 as 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 as shown in SEQ ID NO.10.

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

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

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

[0028] The molecular marker Ks4 is a nucleotide sequence containing a polymorphism of G / C at the 100th position of the sequence as shown in SEQ ID NO.17; the molecular marker Ks6 is a nucleotide sequence containing a polymorphism of A / G at the 100th position of the sequence as 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 as 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 as shown in SEQ ID NO.20.

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

[0031] 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; and the stalk rot is preferably caused by Fusarium graminearum.

[0032] The molecular marker Ks6 has a genotype of AA at the polymorphic site, 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.

[0033] The above SNP molecular markers are closely linked to the maize stalk rot resistance QTL locus qGSR10.06.

[0034] Specifically, the molecular marker Ks4 is obtained by PCR amplification using a primer pair with sequences as shown in SEQ ID NO.3-5 and 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 as shown in SEQ ID NO.6-8 with 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 of KASP markers 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; a genotype of CC, 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 genotype of the polymorphic site as AA, corresponding to a disease-resistant gene; the genotype is 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 markers.

[0042] As an embodiment of the present invention, the primers include primers shown as SEQ ID NO.3-5, and / or primers shown as SEQ ID NO.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 closely linked to the QTL or a SNP molecular marker associated with corn stalk rot resistance or the primer or the reagent or 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 of molecular marker-assisted breeding for maize resistance to stalk rot;

[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 a corn stalk rot resistance phenotype, comprising the following steps:

[0056] (1) extracting genomic DNA of corn to be identified;

[0057] (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;

[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 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 making the corn chromosome 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) A first corn plant is provided, 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

[0069] 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;

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

[0071] (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);

[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 their progeny plants having increased resistance to stalk rot obtained by the above method belong to the protection scope 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) Isolating 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 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

[0079] 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.

[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 position 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 plant or its progeny plant produced by the above method belongs to the protection scope 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, or a nucleotide sequence corresponding to the 60th base of the sequence shown in SEQ ID NO.9, or a nucleotide sequence corresponding to the 70th base of the sequence shown in SEQ ID NO.11, or a nucleotide sequence corresponding to the 80th base of the sequence shown in SEQ ID NO.13, or a nucleotide sequence corresponding to the 90th base of the sequence shown in SEQ ID NO.15, or a nucleotide sequence corresponding to the 100th base of the sequence shown in SEQ ID NO.17, or 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 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;

[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 corn 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 closely linked to the QTL locus.

[0090] The present invention has found through a large number of tests that, in the identification or screening of corn stalk rot resistance using the above molecular markers, the accuracy of identifying stalk rot resistance when Ks4 is used alone is about 58%; the accuracy of identifying stalk rot resistance when Ks6 is used alone is about 50%; when both are used for detection, the accuracy of identifying stalk rot resistance is 84%. This indicates that the two SNP molecular markers are effective in assisting the selection of corn stalk rot resistance phenotypes, and further indicates that the identification of corn stalk rot resistance can be achieved by using molecular markers Ks4 and Ks6 alone, and the combined use of molecular markers Ks4 and Ks6 can significantly improve the accuracy of identifying corn stalk rot resistance.

[0091] The SNP site in the present invention has a clear position, a convenient and rapid detection method, is not affected by the environment, has a stronger purpose, a small workload, a higher efficiency, and a low cost. Therefore, by detecting the SNP site, identification and auxiliary screening can be performed in the seedling stage, greatly saving production costs and improving selection efficiency. In corn breeding, the molecular markers of the present invention and the detection method thereof 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; wherein A is the field phenotype of stem rot of the parental inbred lines (HR: disease resistance, HS: disease susceptible); B is the observation of stem rot occurrence by splitting 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 of stem rot; *** indicates significance 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 3This is the QTL positioning of the disease classification of Fusarium graminearum stem rot under single environment and multi-environment joint analysis conditions in Example 3 of the present invention. In the figure, the lines represent the QTL positioning results of Fusarium graminearum stem rot-related traits on chromosome 10 of maize in 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 QTL, the lines represent the additive effect values ​​of different QTLs; the X-axis represents the genetic map position in cM; the Y-axis represents the additive effect value, and the positive and negative values ​​represent the direction of action.

[0095] Figure 4 Schematic diagram of effect verification and fine positioning of qGSR10.06 in Example 3 of the present invention: Figure A is the effect verification of qGSR10.06; Figure B is the fine positioning of qGSR10.06; Figure C is the DSI distribution of heterozygous individual segregating offspring in fine positioning. The recombinant offspring verification method was used to verify the QTL effect and fine positioning, and the t test was used to analyze whether there was a difference in DSI between the homozygous offspring of the disease-resistant allele and the homozygous offspring of the disease-susceptible allele of each recombinant individual. If there is a significant difference in DSI between the two groups of genotypes (P<0.05), it is inferred that the parental recombinant individual carries the disease-resistant gene, 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 individual does not carry the disease-resistant gene, which is represented by S. The black rectangle represents the homozygous disease-resistant allele segment; the white rectangle represents the homozygous disease-susceptible allele segment; the gray rectangle represents the heterozygous allele segment. Molecular markers are markers used when analyzing the results. 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

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

[0097] KA105 and KB204 used in the following examples are inbred lines independently bred by the corn 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 group and Shaanxi B group under different density conditions [J]. Acta Agronomica Sinica, 2017, 43(9): 1328-1336.). The seeds of the parental inbred lines of the test materials were provided by the corn breeding team of the College of Agriculture of Northwest Agriculture and Forestry University.

[0098] The Fusarium graminearum strain was provided by Associate Researcher Long Shusheng from the College of Plant Protection, Northwest A&F 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 stalk rot resistance traits

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

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

[0102]

[0103]

[0104] 2. Field design

[0105] The parents KA105 and KB204 and their 240 RIL populations were tested for stem rot resistance in Yangling, Shaanxi and Sanyuan, Shaanxi from 2019 to 2020. The location and year were uniformly defined as the environment, abbreviated as "year + location initials", such as 2019YL. The field experiment adopted an incomplete randomized block design, with 2 replications per environment, single-row planting, 4m row length, 0.6m row spacing, and a density of 5500 plants / mu. The field management measures were the same as the local field corn production management.

[0106] 3. Pathogen culture

[0107] Propagation of Fusarium graminearum: Inoculate the pathogenic bacteria strain isolated from a single spore onto PDA (Potato Dextrose Agar) medium and culture it in a constant temperature incubator at 25°C for 5-7 days in the dark. When the mycelium covers the surface of the medium, cut it into equal parts together with the medium and inoculate it onto sterilized corn kernel medium to propagate the Fusarium graminearum inoculum.

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

[0109] Propagation of Fusarium graminearum inoculated materials: Select healthy and plump corn kernels and soak them in water for 20h-22h, boil them in boiling water for 100min, lay the cooked corn kernels flat and dry them; pack them into high temperature resistant inoculation bags of 500g each, sterilize them at 121℃ and autoclave for 50min; after cooling, inoculate the grown Fusarium graminearum on PDA culture medium and seal them with breathable sealing film; culture them in the dark at 25℃ for 15-20 days until the mycelium covers the surface of the corn kernels. During this period, rub the bag every two days to make the corn kernels and the fungus blocks fully contact. Dry the grown inoculated materials in the shade for 3-4 days, put them into sacks and put them in the cold storage for use.

[0110] 4. Intra-field bacteria

[0111] During the tasseling period of corn, the field inoculation method of burying the roots is used. Before inoculation, the cultivated seed inoculation material is stirred and mixed. During inoculation, a pit is dug 5-10 cm near the root of the plant, some fibrous roots are cut off to create wounds, 75-85 infected corn kernels are placed in the pit, and the soil is covered and compacted. After the inoculation is completed, the field is irrigated to keep the soil moist to promote the reproduction and infection of pathogens.

[0112] 5. Investigation of stem rot resistance traits

[0113] 50–55 days after inoculation, the occurrence of corn stalk rot in the field was identified by the stem splitting method. The plants were cut at the cob attachment point and split longitudinally along the stem to observe the pathogen infection at the root and stem base and the degree of pith necrosis. The disease rating scale (DRS) was used to identify the phenotype of stalk rot, which was divided into 5 levels:

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

[0115] Level 3: disease-resistant, the plant 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 medullary tissue between the first and second internodes at the base of the stem shrinks and decomposes, and browning symptoms appear;

[0117] Level 7: Infected, 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, and 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 stem of the plant 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] Select 10 plants with the same growth in each family, observe and record the disease grade of stem rot one by one, and use the average value as the phenotypic value of each family. Investigate and record the number of days from sowing to 50% of the plants shedding pollen in each family.

[0120] 6. Phenotypic data analysis

[0121] 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" program in SAS (V9.2). The mixed linear model was: y = μ + gi + ej + ε, where μ represents the total mean of the phenotype, gi is the genotype effect of the 'i'th family, ej is the effect of the 'j'th environment, and ε is the random residual term. Genotype was used as a fixed effect, flowering date as a covariate, environment, replication within environment, and the interaction between genotype and environment as random effects, and the mixed linear model was used to calculate the variance components.

[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 effect of environmental variation on phenotypes, the SAS (V9.2) mixed linear model was used to estimate the best linear unbiased prediction (BLUP) of each family, and the BLUP value was used as the data for multi-environment joint analysis to draw phenotypic distribution histograms and multi-environment joint QTL positioning.

[0123] Identification of resistance to Fusarium graminearum stem rot in recombinant inbred lines and parental materials under different environments Figure 1 ), the statistical analysis results showed that there were extremely significant differences in the disease grading between parental materials in different groups (Table 3).

[0124] Table 3

[0125]

[0126] Note: 2019YL: Yangling in 2019; 2019SY: Sanyuan in 2019; 2020YL: Yangling in 2020; 2020SY: Sanyuan in 2020; BLUP: multi-environment joint analysis.

[0127] In the RIL population, disease grading showed wide and continuous variation between 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 grading was 1.00-9.00, the mean was between 2.91-5.43, and the coefficient of variation was 33.27%-65.13%. After calculating the BLUP value to eliminate the environmental influence, the coefficient of variation of disease grading in the population was reduced to 33.26%. Disease grading in a single environment showed a skewed normal distribution ( Figure 2 ), the BLUP values ​​of joint analysis of multiple environments were normally distributed.

[0128] Table 4 Variance and heritability analysis of stem rot phenotypic indicators in multiple environments

[0129]

[0130] Note: *** indicates P < 0.001.

[0131] The results of the joint variance analysis showed (Table 4) that the differences between genotypes in the mapping population were extremely significant, and the differences between environments and the interaction between genotype and environment were extremely significant. The estimated genetic variance was greater than the estimated variance of the environment and the interaction between genotype and environment, indicating that genetic factors were the main source of variation and were greatly affected by the environment. The heritability of stem rot resistance in the recombinant inbred line population was 83.23%.

[0132] Example 2 Maize linkage map construction

[0133] At the 5-6 leaf stage of corn, 5 young leaves of each family with consistent growth in the RIL population in Example 1 were selected and mixed, and genomic DNA was extracted using the conventional CTAB method. Genotype data were obtained by sequencing 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 Beijing Academy of Agricultural and Forestry Sciences. The linkage map was constructed using QTL ICImapping 4.2 software. The ".map" function was used to construct a linkage map, and the Kosambi function was selected to estimate the genetic distance. Finally, a total of 48,087 polymorphic SNP markers were screened and merged into 7,200 bin markers. A high-density linkage map was constructed using 240 families combined with 7,200 bin markers. The total length of the map is 16,501cM, and the average genetic distance between adjacent markers is 2.29cM.

[0134] Example 3: QTL location and linked SNP site mining for maize resistance to stalk rot

[0135] 1. QTL positioning

[0136] Based on the linkage map constructed in Example 2, the phenotypic values ​​of disease grading in the RIL population in single environment and multi-environment joint analysis were combined to detect QTL for resistance to Fusarium graminearum stem rot. The composite interval mapping method (CIM) of Windows QTL Cartographer V2.5 software was used for initial positioning and genetic effect analysis. The LOD threshold was set to 3, and the confidence interval of the target QTL was determined by decreasing 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.

[0137] Using the phenotypic data of disease classification in single environment and multi-environment joint analysis, WinQTLCart V 2.5 Composite interval mapping was used to locate QTLs for resistance to Fusarium graminearum stem rot in maize.

[0138] In the mapping population, a resistance QTL was detected on chromosome 10 bin 10.06 ( Figure 3 ), named qGSR10.06, with a physical position between 137,771,944bp–143,942,505bp (B73 AGP_V3), and a confidence interval of approximately 6.17Mb. qGSR10.06 co-localized with 2020SY and BLUP values ​​(Table 5), with LOD values ​​between 4.25 and 7.06, and explained phenotypic variation of 7.78%-8.64%. The resistance allele originated from the disease-resistant parent KA105.

[0139] Table 5 QTL analysis of disease classification of Fusarium graminearum stem rot under single and multi-environment combined conditions

[0140]

[0141] 2. Verification and fine positioning of QTL qGSR10.06 effect

[0142] The QTL effect verification and fine positioning were carried out by the recombinant progeny verification method. According to the initial QTL positioning results, the remaining heterozygous lines with fixed background genotypes in the target segment were screened in the RIL population, and the KASP marker was developed to screen the 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 classification 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 by a one-tailed t test. If the difference between different genotypes was significant (P < 0.05), it means that the major effect QTL is located in the heterozygous region, and vice versa.

[0143] DSI (%) = Σ (disease grade × number of plants with corresponding grade) × 100 / (maximum disease grade × total number of plants with identified stem rot phenotype)

[0144] One remaining heterozygous line material 638RIL-17-1 was screened out from the recombinant inbred line population, which was heterozygous in the qGSR10.06 segment. The same method was used to verify the effect of qGSR10.06, and the results showed that the homozygous allele from the disease-resistant parent could significantly improve the field resistance to stem rot (P<0.001)( Figure 4 Figure A). Among them, the average DSI of the homozygous disease-resistant KA105 / KA105 genotype plants in Yangling in 2021 was 23.46%, and 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%, and 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, the effect of qGSR10.06 was detected, and the results of the two-year test were significantly different, 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 field resistance of plants to Fusarium graminearum stem rot.

[0145] Molecular markers were encrypted in the candidate interval of qGSR10.06, and molecular marker primers were designed with reference to the V3 version sequence of the maize B73 reference genome. The molecular markers Ks4 and Ks6 at both ends of the candidate interval, Ks4 is located at position 142279908 of maize chromosome 10, and the mutant base is G or C; Ks6 is located at position 143572421 of maize chromosome 10, and the mutant base is A or G. The heterozygous offspring produced by 638RIL-17-1 were divided into different recombinant types. The results of fine mapping of qGSR10.06 showed that there was a significant difference in DSI between plants carrying the homozygous KA105 genotype and the homozygous KB204 genotype in types II, III, IV and V (P<0.05), while there was no significant difference in DSI between plants carrying the homozygous genotypes in types I and VI (P>0.05). Therefore, it was concluded that the KA105 donor fragment in the overlapping heterozygous segments of types II, III, IV and V carried qGSR10.06 ( Figure 4 Figure B). Based on whether each type carries qGSR10.06, it can be inferred that the site is located between markers Ks4 and Ks6 in the heterozygous interval of types I and IV, with a physical distance of about 1.29Mb (B73AGP_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 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.

[0146] 3. KASP molecular marker development

[0147] The polymorphic SNP located in the target QTL segment was selected to develop KASP markers for effect verification. The design parameters of KASP marker primers are as follows: primer length is 20-27bp, GC content is between 40%-60%, and annealing temperature is between 59℃-65℃ (optimum temperature is 63℃). The designed primers are shown in Table 1. For application in KASP technology, a fluorescent linker sequence is 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'. The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The primer sequences used to amplify Ks4 in this example are shown in SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.5, respectively; the primer sequences used to amplify Ks6 are shown in SEQ ID NO.25, SEQ ID NO.26, and SEQ ID NO.8, respectively.

[0148] The PCR reaction system is as follows: the total system is 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 mixture. The preferred primer mixture ratio is: 12 μL (100 μM) each of forward primer PrimerX and Primer Y, 30 μL reverse primer Primer R (100 μM), and 46 μL ddH2O.

[0149] The PCR reaction program was as follows: pre-denaturation at 94°C for 15 min, first step amplification reaction, denaturation at 94°C for 20 s, annealing at 61°C for 60 s, set for 10 cycles, second step amplification reaction, denaturation at 94°C for 20 s, annealing and extension at 55°C for 60 s, set for 38 cycles.

[0150] The PCR reaction products were scanned and the fluorescence data were read using a FLUOstar Omega microplate reader (BMG Labtech, Offenburg, Germany). The SNP typing software KclusterCaller (V 3.4.1.36; LGC Hoddesdon, UK) was used to read the genotype data and perform cluster analysis. 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.

[0151] If the genotype at the KASP molecular marker site Ks4 of the maize to be tested is GG, the genotype of qGSR10.06 is a disease-resistant gene; if the genotype at the KASP molecular marker site Ks4 of the maize to be tested is CC, the genotype of qGSR10.06 is a disease-susceptible gene.

[0152] If the genotype at the KASP molecular marker site Ks6 of the corn to be tested is AA, the genotype of qGSR10.06 is a disease-resistant gene; if the genotype at the KASP molecular marker site Ks6 of the corn to be tested is GG, the genotype of qGSR10.06 is a disease-susceptible gene.

[0153] Example 4 Application of QTL qGSR10.06 and tightly linked SNP molecular markers in identification, screening and breeding of corn with resistance to stalk rot

[0154] The material for detecting the polymorphism or genotype of the SNP molecular marker site closely linked to the QTL of corn Fusarium graminearum stem rot resistance of the present invention has at least the following applications:

[0155] (1) Identify or assist in identifying corn resistant to stalk rot;

[0156] (2) preparing related products for identifying or assisting in identifying corn resistant to stalk rot;

[0157] (3) Maize assisted breeding or preparation of products for maize assisted breeding.

[0158] The method for identifying or assisting in identifying resistance to maize stalk rot (Fusarium graminearum) using molecular markers closely linked to maize stalk rot resistance QTLs is as follows:

[0159] If the genotype of the molecular marker Ks4 in the tested corn is GG, the genotype of qGSR10.06 is a disease-resistant gene; if the genotype of the molecular marker Ks4 in the tested corn is CC, the genotype of qGSR10.06 is a disease-susceptible gene;

[0160] If the genotype of the molecular marker Ks6 in the tested corn is AA, the genotype of qGSR10.06 is a disease-resistant gene; if the genotype of the molecular marker Ks6 in the tested corn is GG, the genotype of qGSR10.06 is a disease-susceptible gene;

[0161] 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.

[0162] The SNP molecular marker developed in Example 3 was used to screen maize stalk rot resistant materials. The test materials were 239 recombinant inbred families in the recombinant inbred line population except the qGSR10.06 fine positioning family 638RIL-17-1. 74 recombinant inbred lines were screened to be homozygous genes at the Ks4 and Ks6 molecular markers. The stalk rot phenotype was identified. It was found that when the genotypes of the molecular markers Ks4 and Ks6 were both homozygous disease resistance alleles GG and AA, 38 of the 43 recombinant inbred lines showed high resistance to stalk rot, with disease grading between 2.42 and 3.92, and the accuracy rate of stalk rot resistance identification was 88%;

[0163] When only the molecular marker Ks4 was homozygous for the disease resistance allele type GG, 10 of the 14 recombinant inbred lines showed resistance to stem rot, with disease grade ranging from 1.96 to 4.41, and the accuracy of stem rot resistance identification was 71%;

[0164] 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 the accuracy of stem rot resistance identification was 63%;

[0165] When the genotypes of molecular markers Ks4 and Ks6 were both homozygous susceptible alleles CC and GG, 28 of the 38 recombinant inbred lines were susceptible to stem rot, with disease grade of 5.18-7.86, and the accuracy rate of stem rot susceptible identification was 74%.

[0166] Table 6 Alleles and phenotypes at marker qGSR10.06

[0167]

[0168]

[0169]

[0170]

[0171] Note: Ks4: GG (disease resistance allele), CC (disease susceptible allele); Ks6: AA (disease resistance allele), GG (disease susceptible allele)

[0172] Table 7 Accuracy of identification of stem rot resistance using qGSR10.06 marker

[0173]

[0174] Note: In the estimation of the accuracy of stem rot resistance identification, the families with high resistance and disease resistance of the recombinant inbred lines 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)

[0175] The molecular markers at both ends of the candidate interval of qGSR10.06 are Ks4 and Ks6. When Ks4 is used alone, the accuracy of stem rot resistance identification is about 71%; when Ks6 is used alone, the accuracy of stem rot resistance identification is about 63%; when both are used for detection, the accuracy of stem rot resistance identification is 88%. This shows that the two SNP molecular markers developed by the present invention are effective for auxiliary selection of corn stem rot resistance phenotypes, and can be used alone or in combination for early identification or auxiliary identification of corn stem rot resistance phenotypes, which can greatly save production costs, improve selection efficiency, and accelerate the corn breeding process.

[0176] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

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 B73AGP_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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