Method for accurately identifying fusarium ear rot resistance phenotype of maize through adaptive gene mining

Abstract

The invention belongs to the field of maize disease resistance identification and gene mining, and particularly relates to a maize fusarium ear rot resistance phenotype accurate identification method for adaptive gene mining, which comprises the following steps: S1, setting a resistance gene positioning identification garden, including a preliminary positioning identification garden and a fine positioning identification garden, selecting a land parcel which is flat in terrain, uniform in fertility, perfect in irrigation and drainage facilities, free of shading and prone to diseases as an identification nursery; s2, selecting a positioning population, adopting an F23 character segregation population and the like as a preliminary positioning identification nursery, and adopting an F3 population, an RIL population, a BC population and an RNA-SEQ detection material as a fine positioning identification nursery; and S3, performing field test design of the identification nursery. According to the method, accurate identification of the corn fusarium ear rot resistance phenotype is achieved by standardizing identification garden setting, optimizing inoculation and investigation processes and linking a molecular technology, reliable phenotype data support is provided for efficient positioning, cloning and related research of anti-disease genes, and corn anti-disease molecular breeding is assisted.

Description

Technical Field

[0001] This invention relates to the field of maize disease resistance identification and gene mining technology, and in particular to a method for accurate identification of maize resistance to Fusarium ear rot phenotype through adaptive gene mining. Background Technology

[0002] Maize Fusarium ear rot is an important disease caused by Fusarium fungi (such as Fusarium graminearum and Fusarium verticillatum), which can lead to ear rot and reduced kernel quality, seriously affecting maize yield and economic benefits. Breeding and planting disease-resistant varieties is the most economical and effective measure to control this disease, and the discovery of disease-resistant genes is the foundation of disease-resistant breeding.

[0003] Accurate phenotypic identification is a prerequisite for gene mining. However, current methods for identifying maize resistance to Fusarium ear rot phenotypes suffer from problems such as non-standardized identification nursery setup, inconsistent inoculation methods, incomplete phenotypic survey indicators, and insufficient integration with gene mining technology. These issues result in poor accuracy and repeatability of identification results, making it difficult to meet the needs for precise localization and cloning of resistance genes. Therefore, we propose a precise phenotypic identification method for maize resistance to Fusarium ear rot that is adapted to gene mining to address the above problems. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a precise identification method for maize resistance to Fusarium ear rot phenotype based on adaptor gene mining.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A precise identification method for maize resistance to Fusarium ear rot phenotype based on adaptor gene mining includes the following steps:

[0007] S1: Set up resistance gene localization identification nurseries, including preliminary localization identification nurseries and fine localization identification nurseries. Select plots with flat terrain, uniform fertility, complete irrigation and drainage facilities, no shade and easy disease occurrence as identification nurseries.

[0008] S2: Select the target population, and use F for preliminary location identification. 23 Segregating populations with equal traits, the fine-positioning identification garden uses F3 population, RIL population, BC population and RNA-SEQ detection materials;

[0009] S3: Conduct field trials for identification, with different planting methods for different populations, including row length, row spacing, number of seedlings per row, and control settings;

[0010] S4: Field management of the identification nursery, including watering, fertilization, and pest control, without spraying fungicides, and watering the inoculation material at specific times to promote the spread of pathogens;

[0011] S5: Prepare Fusarium inoculum, and culture and propagate Fusarium graminearum and Fusarium pseudoverticum using specific culture media, and adjust the concentration of spore suspension;

[0012] S6: During specific growth stages of maize, different Fusarium species are inoculated using the silk channel method or the ear needle pricking method, and then moisturized.

[0013] S7: In the late milk stage of maize kernels, investigate the disease resistance phenotypes such as ear filling, resistance to infection, resistance to spread, and disease incidence on the cob.

[0014] S8: Collect plant samples from different populations and extract DNA or RNA;

[0015] S9: Disease resistance genes were located using methods such as BSA, genetic mapping, and GWAS. Candidate genes were screened by combining transcriptome data analysis, and then cloned and validated.

[0016] Preferably, in step three, the identification materials for the preliminary identification nursery are planted in a randomized block arrangement in the field, with a row length of 4.8m, a row spacing of 0.6m, and 25 seedlings per row; parental materials P1, P2 and their F1 are planted in 4 rows each, and F2 is planted in 30 rows; control 1 is a combination of disease-resistant parents and disease-susceptible parents, and control 2 is a combination of inbred line BT resistant to ear rot and inbred line B73 susceptible to ear rot, with two replicates of each of the two control materials.

[0017] Preferably, in step five, during the preparation of Fusarium graminearum inoculum, a highly pathogenic Fusarium graminearum strain is inoculated onto a PDA medium plate and cultured at 28°C for approximately 7 days. Mycelial blocks are then picked and cultured in CMC liquid medium at 25°C in the dark with shaking for 1–2 weeks. After filtration, the culture is stored at 4°C. At the time of inoculation, the spore suspension concentration is adjusted to 2 × 10⁻⁶. 5 spores / mL.

[0018] Preferably, in step six, for Fusarium graminearum ear rot, 5–7 days after artificial self-pollination, the inoculation method is the silk channel injection method, with 2 mL of spore suspension injected into each ear, and 1 mL of pollination identification material is inoculated; for Fusarium verticillatum ear rot, 12 days after corn silking, the inoculation method is the ear needle prick method, with 2 mL of inoculum injected into each ear, and 1 mL of pollination identification material is inoculated.

[0019] Preferably, in step seven, the fruit set rate is recorded as 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. If the fruit set rate is less than 70%, the disease data of that fruit ear is removed. The resistance to infection is recorded as Y / N, i.e., diseased / no diseased. The resistance to spread includes visually observing the relative diseased area of ​​the fruit ear and measuring the length of the fruit ear and the length of the lesion spread.

[0020] Preferably, in step eight, before inoculation, F2 population sampling involves tagging individual plants and drying and preserving leaves approximately 15 cm in length; RNA-SEQ detection materials are collected at 0, 6, 12, 24, 48, 72, and 240 hours after inoculation, with 10 grains per spike, and 3 plants are mixed as one replicate, for a total of 3 replicates, which are then flash-frozen in liquid nitrogen and stored at -80°C.

[0021] Preferably, in step nine, the BSA analysis used for disease resistance gene localization includes the following steps: fastqc data quality control, BWA alignment to the reference genome, samtools processing of sam / bam files, picard deduplication, GATK variant detection, bcftools indexing, and vcftools filtering.

[0022] Preferably, in step nine, the verification of disease-resistant genes employs techniques such as gene knockout, silencing, transgenic overexpression, and genetic complementation.

[0023] The beneficial effects of this invention are:

[0024] 1. By standardizing the setup of the identification nursery, different needs such as preliminary positioning and fine positioning can be distinguished, ensuring the consistency and comparability of the planting of experimental materials and providing a stable field environment for phenotypic identification.

[0025] 2. The preparation and inoculation methods of Fusarium inoculum have been optimized. Dedicated procedures have been designed for different Fusarium species, such as Fusarium graminearum and Fusarium verticillatum, which has improved the uniformity and controllability of disease development and ensured the significance of phenotypic differences.

[0026] 3. The phenotypic survey indicators have been refined to cover seed setting, resistance to infection, resistance to spread, and ear disease, comprehensively reflecting the phenotypic characteristics of maize resistance to Fusarium ear rot and providing multi-dimensional data for gene mapping.

[0027] 4. It integrates molecular biology techniques such as BSA, RNA-seq, and gene cloning, achieving seamless integration of phenotypic identification and gene mining, and improving the efficiency and accuracy of disease resistance gene localization and cloning. Attached Figure Description

[0028] Figure 1 This is a comparison chart of the estimated diseased area of ​​corn ears (A) and the actual diseased area of ​​corn ears (B) according to the present invention;

[0029] Figure 2 This is a field randomized block arrangement planting diagram of the present invention;

[0030] Figure 3 The planting diagram shows the F2 harvested ears of this invention arranged in order from resistant to susceptible according to the phenotypic identification results.

[0031] Figure 4This invention demonstrates how, in the absence of molecular markers, highly resistant plants are screened through field phenotypic identification (highly susceptible plants are selected when locating the susceptibility gene) and then harvested and planted by single ear.

[0032] Figure 5 This is a diagram of the RIL population planting in this invention. Detailed Implementation

[0033] The following is in conjunction with the appendix Figures 1-5 This application will be described in further detail.

[0034] This application discloses a method for precise identification of maize resistance to Fusarium ear rot phenotype based on adaptor gene mining.

[0035] Reference Figures 1-5 A precise identification method for maize resistance to Fusarium ear rot phenotype based on gene mining includes the following steps:

[0036] S1: Setting up a resistance gene localization and identification nursery

[0037] Preliminary identification nursery: Select a flat, fertile plot with complete irrigation and drainage facilities, no shade, and prone to disease. Identification materials were planted in a randomized block arrangement in the field, with a row length of 4.8m, a row spacing of 0.6m, and 25 seedlings per row. Parental materials P1, P2, and their F1 strains were planted in 4 rows each, and F2 strains in 30 rows. Control 1 consisted of a combination of disease-resistant and disease-susceptible parents; Control 2 consisted of the inbred line BT (highly resistant, HR) and the inbred line B73 (highly susceptible, HS) for ear rot resistance. Two replicates were planted for each control group.

[0038] Precise location identification plot: Select plots that meet the above conditions.

[0039] F3 population: Single ears harvested from F2 were planted in order of resistance to susceptibility based on phenotypic identification results, with a planting row length of 4.8m and a row spacing of 0.6m, leaving 25 seedlings per row. The first 5 seedlings of each ear row were used for self-pollination (to construct the RIL population), and the last 20 seedlings were used for gene mapping. Parental materials P1 and P2 were each planted in 4 rows. The control setup was the same as the preliminary mapping identification nursery.

[0040] BC population: Before obtaining molecular markers, highly resistant plants were selected through field phenotypic identification (highly susceptible plants were selected when locating the susceptibility gene), and harvested and planted as single ears. Planting rows were 3m long, with a row spacing of 0.6m, and 15 seedlings were retained per row. Each plant was backcrossed with a susceptible parent. After obtaining molecular markers, resistant plants were selected through molecular marker identification (susceptible plants were selected when locating the susceptibility gene), and harvested and planted as single ears (planting method as above). Each plant was backcrossed with a susceptible parent (resistant parents were selected when locating the susceptibility gene).

[0041] RIL group: F 23After the initial population, the first 5 plants of each row of individual ears in each generation were used for self-pollination and seed saving. From each row of 5 ears, the plumpest single ears were randomly selected for threshing and used for the next generation. The planting rows were 3m long and 0.6m apart, with 15 seedlings per row. The disease-resistant parent and the disease-susceptible parent served as controls.

[0042] RNA-SEQ detection materials: For each sampling time point (0, 6, 12, 24, 48, 72 and 240h), two rows of both resistant and susceptible parental lines were planted, for a total of 14 rows, with 25 plants per row. The planting rows were 4.8m long and 0.6m apart.

[0043] S2: Identification of field management

[0044] Management was carried out according to the requirements of the breeding station's routine experimental fields, including watering, fertilization, and pest control, but without the application of fungicides. A relatively high-intensity watering was applied to the inoculated material during the later stages of grain filling to promote the spread of pathogens in the corn ears and the onset of disease.

[0045] S3: Preparation of Fusarium inoculum

[0046] Preparation of Fusarium graminearum inoculum: Inoculate highly pathogenic Fusarium graminearum strains onto PDA agar plates and incubate at 28°C for approximately 7 days. Using an inoculation needle, pick three 6mm mycelial blocks and place them in 200mL CMC liquid medium (formula: 1.0g NH4NO3, 1.0g KH2PO4, 0.5g MgSO4·7H2O, 1.0g yeast extract, 15.0g carboxymethyl cellulose, 1L distilled water; dispense 200mL into each Erlenmeyer flask and sterilize at 121°C for 20min). Incubate in the dark with shaking at 25°C for 1–2 weeks to produce a large number of large conidia. Filter the liquid through double-layered sterile gauze and store at 4°C. When preparing for inoculation, adjust the spore suspension concentration to 2×10⁻⁶. 5 One spore per mL is sufficient for inoculation.

[0047] Preparation of Fusarium oxysporum inoculum: Inoculate Fusarium oxysporum onto PDA medium plates and incubate at 28°C for about 7 days until the mycelium fully colonizes the plate. Store at 4°C for later use.

[0048] Method 1: 10-15 days before inoculation, take an appropriate amount of healthy, plump corn kernels, boil them in boiling water for 30 minutes, place them in Erlenmeyer flasks, and autoclave at 121℃ for 60 minutes. Inoculate the culture medium grown on the plate into the Erlenmeyer flasks, seal the flasks, and incubate at room temperature for 15 days. One day before inoculation, prepare a solution of 5×10⁻⁶ microbial culture medium with sterile distilled water. 6 A spore suspension of 1 spore / mL was prepared. Tween-80, a surfactant, was added to the spore suspension at a concentration of 2 μL / mL, and the mixture was thoroughly mixed.

[0049] Method 2: Inoculate a highly pathogenic *Fusarium verticillatum* strain into a liquid culture medium containing mung bean soup or pea soup. Incubate in the dark with shaking at 25°C for 4–5 days to produce a large number of conidia. Store at 4°C. When ready for inoculation, filter the liquid through four layers of sterile gauze and adjust the stock solution to a concentration of 5 × 10⁶ spores / mL with sterile purified water. This solution is then ready for inoculation.

[0050] S4: Inoculation of pathogens

[0051] For Fusarium wilt of cereal ears: Inoculate 5–7 days after artificial self-pollination to ensure normal ear setting. Use the filament channel injection method, inserting a quantitative continuous syringe into the hollow part of the filament channel on the side of the ear, injecting 2 mL of spore suspension per ear, and 1 mL for pollination identification materials. After injection, spray the entire field with water to maintain moisture, and spray again in the evening or morning 12 hours later.

[0052] Fusarium wilt of the ear: The optimal inoculation time is 12 days after silking of corn. Use a syringe to inject the prepared spore suspension into the middle of the corn ear (the position should be just inside the cob, and the inoculation position of all ears should be consistent). The inoculation volume for each ear is 2 mL, and 1 mL for pollination identification materials. After inoculation, gently pinch the wound with your fingers to prevent the bacterial solution from flowing out.

[0053] S5: Disease Resistance Phenotypic Survey

[0054] The investigation was conducted during the late milk-ripe stage of corn kernels, focusing on the ears of the plant.

[0055] Ear filling rate: After removing the husks, observe whether the grain filling is normal to determine if the resistance phenotype has been affected. Record 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. If the filling rate is below 70%, the disease data for that ear should be removed.

[0056] Resistance to infection: Visually inspect the silks, grains, and rachis for any visible disease and fungal hyphae, and record the result as Y / N (with disease / without disease).

[0057] Resistance to spread: Visually assess the relative diseased area of ​​the ear and record it as 0%, 2% (only 1-2 kernels infected), 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%; measure and record the ear length and lesion spread length with a ruler, recording the length as 0cm, 1cm, 2cm, 3cm, 4cm, 5cm, 6cm, 7cm, 8cm, 9cm, 10cm...

[0058] Disease status of the rachis: After removing one row of grains at the inoculation point, visually inspect the rachis for disease and the length of the disease. For severely diseased rachis, it is also necessary to assess the relative length of the disease (if the rachis is diseased, the length of the diseased rachis should be recorded as 0cm, 1cm, 2cm, 3cm, 4cm, 5cm, 6cm, 7cm, 8cm, 9cm, 10cm, etc.).

[0059] S6: Plant Sample Collection

[0060] F2 population sampling: Before inoculation at the seedling stage, individual plants in the F2 generation were tagged. Before sampling, the plant number was marked on the 3rd or 4th unfolded leaf. Leaves approximately 15cm in length were taken, and a row of leaves were placed in a 20cm × 10cm mesh bag and dried in a well-ventilated, shady place. The completely dried leaves were then transferred to pollination-grade sulfuric acid paper bags and stored in a well-ventilated, shady place for later use.

[0061] F3 group sampling: Sampling begins from the 6th plant in each row. All seedlings are tagged during the seedling stage. Before sampling, mark the plant number on the 3rd or 4th unfolded leaf. Take leaves approximately 15cm in length and place the leaves of one row into a small 20cm×10cm mesh bag, then dry in a well-ventilated, shady place. Transfer the completely dried leaves to a pollination-grade sulfuric acid paper bag and store in a well-ventilated, shady place until use.

[0062] BC population sampling: Before inoculation, individual plants in the BC population were tagged. Before sampling, the plant number was marked on the 3rd or 4th unfolded leaf. Leaves about 15cm in length were taken and placed in a 20cm×10cm mesh bag. The leaves were dried in a cool, ventilated place. The completely dried leaves were then transferred to a pollination-grade sulfuric acid paper bag and stored in a cool, ventilated place for later use.

[0063] RIL population sampling: The sampling method is the same as that for the BC population.

[0064] RNA-SEQ detection material sampling: The inoculation materials were NIL-qFERgene+ and NIL-qFERgene-. At 0, 6, 12, 24, 48, 72 and 240 h after inoculation, 10 grains were taken from each spikelet, and 3 strains were mixed to form a replicate. A total of 3 replicates were taken (each replicate was divided into two tubes, one tube was sent for sequencing and the other tube was kept as a backup). The samples were flash-frozen in liquid nitrogen and then stored at -80℃.

[0065] S7: DNA / RNA extraction from plant samples

[0066] F 23 DNA extraction from F3, BC, and RIL populations: DNA was extracted using a fully automated nucleic acid extractor and the magnetic bead method. Specific steps included sample collection via punching, sample grinding, addition of lysis buffer in a water bath, centrifugation, transfer of supernatant to square well plates, and extraction using a fully automated nucleic acid extractor according to a specific program.

[0067] DNA extraction from BSA materials: Based on field phenotypic identification results, DNA was extracted from F... 23 Several samples of extremely resistant and extremely susceptible plants were selected, and DNA was extracted from each. Equal amounts of DNA were mixed to construct two gene pools (resistant pool BR and susceptible pool BS), and two parental pools were also constructed (five plants from each parent were selected and their DNA extracted). Specific steps included grinding, adding extraction buffer and water bath, centrifugation, transferring the supernatant, adding reagents for purification, precipitation, washing, and dissolution.

[0068] RNA extraction from RNA-SEQ detection materials: The sample stored at -80℃ was removed and placed on ice blocks. Total RNA was extracted from the sample using the Trizol method or a total RNA extraction kit. The Trizol method includes the following steps: grinding, adding reagents and mixing, centrifugation, aspirating the supernatant, centrifugation again, precipitation, washing, and dissolution.

[0069] S8: Disease Resistance Gene Localization Technology Method

[0070] Commonly used methods for locating disease resistance genes include BSA, genetic mapping, and GWAS.

[0071] Sequencing of disease-resistant / susceptible and disease-resistant / susceptible materials: The material resequencing platform was DNBSEQ; the sequencing length was PE150; and the sequencing depth was 50×.

[0072] BSA analysis includes fastqc (data quality control); BWA (alignment to reference genome); samtools (processing sam / bam files); picard for deduplication; GATK (variant detection); bcftools (indexing); and vcftools (filtering).

[0073] Genetic mapping: The ΔSNP-index method, G'analysis method, and ED method (plotted using R software) were employed.

[0074] Molecular marker development: Based on the variant sites and repetitive sequences within the candidate regions initially located by BSA, Indel markers and SSR markers were developed respectively.

[0075] Precise localization: When the initial candidate region is large, molecular markers are developed for further fine localization, and larger genetic populations or combined with RNA-seq and other methods are used for joint localization.

[0076] Transcriptome data analysis: Differential gene expression analysis was performed using transcriptome data, including FastQC (data quality control); Trimmomatic software (sequence processing); Hisat2 software (alignment to a reference genome); FeaturesCounts software (counting); and DESeq2 software (differential gene expression analysis). Candidate genes were selected based on the results and validated by RT-qPCR.

[0077] S9: Disease-resistant gene cloning technology method:

[0078] Based on the sequencing results of the inbred line DeNovo, the complete sequences of candidate genes were extracted, primers were designed, and the full-length sequences were amplified using high-fidelity Taq polymerase. Simultaneously, total RNA was extracted from both parents and reverse transcribed, and the CDS sequences were amplified by PCR. Specific steps included genomic DNA and total RNA extraction, target gene primer design, PCR amplification, electrophoresis detection, PCR product recovery, sequencing of the recovered products, expression vector construction, transformation of competent cells, single-clone identification, and plasmid extraction.

[0079] In this invention, by standardizing the setting of the identification nursery, different needs such as preliminary positioning and fine positioning can be distinguished, ensuring the consistency and comparability of experimental material planting, providing a stable field environment for phenotypic identification, optimizing the preparation and inoculation methods of Fusarium inoculum, and designing exclusive processes for different Fusarium species such as Fusarium graminearum and Fusarium verticillatum, improving the uniformity and controllability of disease incidence, ensuring the significance of phenotypic differences, refining the phenotypic survey indicators to cover seed setting, resistance to infection, resistance to spread, and ear cob disease, comprehensively reflecting the phenotypic characteristics of maize resistance to Fusarium ear rot, providing multi-dimensional data for gene mapping, and connecting molecular biology techniques such as BSA, RNA-seq, and gene cloning, realizing seamless integration of phenotypic identification and gene mining, and improving the efficiency and accuracy of disease resistance gene mapping and cloning.

[0080] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A method for precise identification of maize resistance to Fusarium ear rot phenotype based on adaptive gene mining, characterized in that, Includes the following steps: S1: Set up resistance gene localization identification nurseries, including preliminary localization identification nurseries and fine localization identification nurseries. Select plots with flat terrain, uniform fertility, complete irrigation and drainage facilities, no shade and easy disease occurrence as identification nurseries. S2: Select the target population, and use F for preliminary location identification. 23 Segregating populations with equal traits, the fine-positioning identification garden uses F3 population, RIL population, BC population and RNA-SEQ detection materials; S3: Conduct field trials for identification, with different planting methods for different populations, including row length, row spacing, number of seedlings per row, and control settings; S4: Field management of the identification nursery, including watering, fertilization, and pest control, without spraying fungicides, and watering the inoculation material at specific times to promote the spread of pathogens; S5: Prepare Fusarium inoculum, and culture and propagate Fusarium graminearum and Fusarium pseudoverticum using specific culture media, and adjust the concentration of spore suspension; S6: During specific growth stages of maize, different Fusarium species are inoculated using the silk channel method or the ear needle pricking method, and then moisturized. S7: In the late milk stage of maize kernels, investigate ear filling, resistance to infection, resistance to spread, and disease incidence on the cob; disease resistance phenotype. S8: Collect plant samples from different populations and extract DNA and RNA; S9: Disease resistance genes were located using BSA, genetic mapping, and GWAS methods. Candidate genes were screened by combining transcriptome data analysis, and then cloned and validated.

2. The method for precise identification of maize resistance to Fusarium ear rot phenotype based on adaptor gene mining according to claim 1, characterized in that, In step three, the identification materials for the preliminary identification nursery were planted in a randomized block arrangement in the field, with a row length of 4.8m, a row spacing of 0.6m, and 25 seedlings per row. The parental materials P1, P2 and their F1 were planted in 4 rows each, and the F2 was planted in 30 rows. Control 1 consisted of a combination of disease-resistant parents and disease-susceptible parents, and control 2 consisted of the inbred line BT resistant to ear rot and the inbred line B73 susceptible to ear rot. Each of the two control groups was planted in duplicate.

3. The method for precise identification of maize resistance to Fusarium ear rot phenotype based on adaptor gene mining according to claim 1, characterized in that, In step five, during the preparation of Fusarium graminearum inoculum, a highly pathogenic Fusarium graminearum strain is inoculated onto PDA medium plates and cultured at 28°C for approximately 7 days. Mycelial blocks are then picked and cultured in CMC liquid medium at 25°C in the dark with shaking for 1–2 weeks. After filtration, the culture is stored at 4°C. At the time of inoculation, the spore suspension concentration is adjusted to 2 × 10⁻⁶. 5 spores / mL.

4. The method for precise identification of maize resistance to Fusarium ear rot phenotype based on adaptor gene mining according to claim 1, characterized in that, In step six, for Fusarium graminearum ear rot, 5–7 days after artificial self-pollination, the inoculation method is the silk channel injection method, with 2 mL of spore suspension injected into each ear, and 1 mL injected into pollination identification materials; for Fusarium verticillatum ear rot, 12 days after corn silking, the inoculation method is the ear needle prick method, with 2 mL of inoculum injected into each ear, and 1 mL injected into pollination identification materials.

5. The method for precise identification of maize resistance to Fusarium ear rot phenotype based on adaptor gene mining according to claim 1, characterized in that, In step seven, the fruit set rate is recorded as 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. If the fruit set rate is lower than 70%, the disease data for that fruit ear is removed. The resistance to infection is recorded as Y / N, i.e., diseased / no diseased. The resistance to spread includes visually observing the relative diseased area of ​​the fruit ear and measuring the length of the fruit ear and the length of the lesion spread.

6. The method for precise identification of maize resistance to Fusarium ear rot phenotype based on adaptor gene mining according to claim 1, characterized in that, In step eight, before inoculation, F2 population sampling involved tagging individual plants and drying and preserving leaves approximately 15 cm in length. RNA-SEQ detection materials were collected at 0, 6, 12, 24, 48, 72, and 240 hours after inoculation, with 10 grains per spike, and 3 plants were mixed as one replicate, for a total of 3 replicates. The samples were then flash-frozen in liquid nitrogen and stored at -80°C.

7. The method for precise identification of maize resistance to Fusarium ear rot phenotype based on adaptor gene mining according to claim 1, characterized in that, In step nine, the BSA analysis used for disease resistance gene localization includes the following steps: fastqc data quality control, BWA alignment to the reference genome, samtools processing of sam / bam files, picard deduplication, GATK variant detection, bcftools indexing, and vcftools filtering.

8. The method for precise identification of maize resistance to Fusarium ear rot phenotype based on adaptor gene mining according to claim 1, characterized in that, In step nine, the verification of disease-resistant genes employs techniques such as gene knockout, silencing, transgenic overexpression, and genetic complementation.

Images