Multi-type maize germplasm resource innovation and hybrid breeding method

Abstract

The application relates to the technical field of crop breeding, and specifically discloses a multi-resistance type maize germplasm resource innovation and hybrid breeding method, which comprises the following steps: pre-evaluating local variety resources; introducing excellent genes of the local varieties into a backbone inbred line for hybridization; performing genotype detection by using molecular markers; performing haploid induction on a hybrid introduction population by using an induction line; doubling the chromosomes of the obtained haploid; simultaneously performing selfing and seed reservation on the obtained DH line and performing combining ability test crossing with a test variety; adjusting the screening weight of the molecular markers according to the correlation analysis result; and performing hybridization on the DH line with the highest rating to form a hybrid. The application adopts differentiated hybridization introduction according to different functional group target properties, and combines feedback iteration of molecular marker continuous screening and combining ability determination results, so that the introduction efficiency and precision of excellent genes of the local varieties into the backbone inbred line are improved.

Description

Technical Field

[0001] This application relates to the field of crop breeding technology, and more specifically, to methods for the innovation of multi-resistant maize germplasm resources and the breeding of hybrid varieties. Background Technology

[0002] Maize is an important food crop, feed source, and industrial raw material in my country. The innovation and utilization of its germplasm resources are the core foundation for breeding breakthrough new varieties. my country has abundant maize germplasm resources. Local varieties have developed unique and excellent traits through long-term natural and artificial selection. They have strong adaptability to local climatic conditions and exhibit rich genetic diversity in terms of stress resistance, quality, and agronomic traits. They are important resources for expanding the genetic basis of maize breeding.

[0003] Improving existing backbone inbred lines using local varieties is an important way to broaden the genetic base of maize germplasm. In existing maize germplasm breeding methods, hybridization and backcrossing are usually used to introduce the superior genes of local varieties into backbone inbred lines. Then, homozygous offspring are obtained through continuous self-pollination or haploid breeding techniques, and molecular marker-assisted selection is used to track and screen for target genes. However, different local varieties carry different superior traits, and the genetic behavior of the genes corresponding to their target traits differs during hybridization and backcrossing. Existing methods often use relatively fixed hybridization schemes for different local varieties, making it difficult to meet the needs of creating multi-resistant germplasm resources in terms of efficiency and accuracy of introducing superior genes of local varieties into backbone inbred lines. Summary of the Invention

[0004] To address the problem that existing maize germplasm breeding methods often result in insufficient efficiency and accuracy in introducing superior genes from local varieties into backbone inbred lines to meet the needs of creating multi-resistant germplasm resources, this application provides a method for the innovation of multi-resistant maize germplasm resources and hybrid breeding.

[0005] The method for innovation of multi-resistant maize germplasm resources and breeding of hybrids provided in this application adopts the following technical solution: Methods for innovation of multi-resistant maize germplasm resources and hybrid breeding include the following steps: S1. Conduct a preliminary assessment of local varietal resources and classify local varieties into different functional groups based on the results of the preliminary assessment; S2. Based on the target traits of different functional groups, design differentiated hybridization introduction schemes to introduce the superior genes of local varieties into the backbone inbred lines for hybridization. S3. In each generation of the hybrid population, genotyping is performed using molecular markers, and individuals carrying the target gene marker are selected based on the test results; S4. Haploid induction is performed on the selected hybrid offspring, and chromosome doubling is performed on the obtained haploids to obtain a homozygous double haploid system, namely the DH line. S5. Simultaneously perform self-pollination and test crosses with the test species to determine the combining ability of each DH line. S6. Perform correlation analysis between the coordination ability determination results and the molecular marker detection results, and adjust the screening weight of the molecular markers based on the correlation analysis results; S7. The DH lines are comprehensively rated based on molecular marker scores, combining ability scores, quality trait scores, and agronomic trait scores. The DH lines with the highest scores are then used for hybridization to complete the breeding process.

[0006] By adopting the above technical solution, and through differentiated hybridization based on target traits of different functional groups, superior genes from different sources are introduced into the backbone lines under various adaptive schemes. Combined with the feedback iteration of molecular marker continuous screening and combining ability determination results from each generation, the screening strategy is continuously optimized. Therefore, the efficiency and accuracy of introducing superior genes from local varieties into backbone inbred lines are improved, solving the problem in existing maize germplasm breeding methods where the efficiency and accuracy of introducing superior genes from local varieties into backbone inbred lines are insufficient to meet the needs of creating multi-resistant germplasm resources.

[0007] Preferably, in step S1, the functional groups include stress-resistant functional groups, quality-oriented functional groups, weather-resistant functional groups, and high-yield functional groups; the local varieties include Longshuiti Old Yellow Corn, Golden Queen, White Horse Tooth, Small Grain Red, White Head Frost, Wild Chicken Red, and Gui Zongxuan.

[0008] By adopting the above technical solutions, and by pre-evaluating local varieties and classifying them into different functional groups, the design of subsequent hybridization introduction programs has a clear functional orientation, avoiding the waste of resources caused by blindly selecting donor materials, and providing a classification basis for differentiated design.

[0009] Preferably, in step S2, the differentiated hybridization introduction scheme is as follows: the stress-resistant functional group and the weather-resistant functional group adopt a 3-hybridization scheme, the quality functional group adopts a 2-hybridization scheme or a 3-hybridization scheme, and the high-yield functional group adopts a 3-hybridization scheme. In each hybridization, 15 to 20 excellent ears are selected from the offspring population according to the breeding target trait, and each hybridization uses backbone lines from different sources but belonging to the same hybrid vigor group as the male parent.

[0010] By adopting the above technical solution, the number of hybridizations is designed differently according to the target traits of different functional groups. This allows the stress-resistant and weather-tolerant functional groups to gradually accumulate beneficial genes through multiple hybridizations, while the quality-type functional groups retain the cumulative effect of quality-related genes through fewer hybridizations. At the same time, each hybridization uses the same heterotic group but different origins of the backbone line as the father, avoiding the homogenization of the genetic basis caused by the continuous use of the same backbone line. This allows the target genes of each functional group to be introduced into the backbone line in an orderly manner under their respective suitable schemes.

[0011] Preferably, in the three-hybridization scheme, the number of female parent plants planted in the first hybridization is not less than 200, and the number of female parent plants planted in the second and third hybridizations is not less than 100 each; the first hybridization of the weather-resistant functional group is sown in a season when the daily maximum temperature is ≥35℃.

[0012] By adopting the above technical solutions, the three-hybrid scheme ensures the diversity of target genes in the population by controlling the planting scale of the maternal population in each generation. At the same time, the first hybridization of the weather-resistant functional group is sown in the high-temperature season, so that the heat-resistant genes are positively selected in the early generations.

[0013] Preferably, in the two-hybridization scheme of the quality-type functional group, after the second hybridization, the grains of the selected single plants are tested for quality traits, and the ears of grains from single plants with a test weight ≥720g / L, crude protein content ≥10%, and crude fat content ≥4% are retained.

[0014] By adopting the above technical solution, the two-hybrid scheme of quality-type functional groups introduces quality trait threshold screening after the second hybridization, so that the ears of single plants that meet the standards for bulk density, crude protein content and crude fat content are retained, and the quality trait-related genes are quantitatively tracked in the hybrid generation.

[0015] Preferably, in step S3, during the screening, 3 to 5 molecular markers are used for screening after the first hybridization, 8 to 12 molecular markers are used for screening after the second hybridization, and 12 to 20 molecular markers are used for verification after the third hybridization. In addition, individuals carrying the top 30% of the target markers are retained during each generation of screening, and ear of fruit with a target gene marker loss rate of more than 30% are eliminated after the third hybridization.

[0016] By adopting the above technical solution, genotyping is performed using molecular markers in each generation after hybridization. A stage-specific marker combination strategy is adopted to increase the screening accuracy of different generations step by step. Combined with the threshold setting of retaining the top 30% of individuals carrying the target marker during each generation screening and eliminating ear of ear with a target gene marker loss rate of more than 30% after the third hybridization, a smooth transition from early protection of genetic diversity to later precise screening is achieved.

[0017] Preferably, in step S4, the identification of haploids includes initial selection using coleoptile color marking, followed by flow cytometry analysis of the ploidy of the haploid kernels obtained in the initial selection, with known diploid maize embryos as a control. Samples with DNA content 0.45 to 0.55 times that of the control are identified as haploids. The chromosome doubling is performed using a root immersion method, in which haploid kernels are soaked in a colchicine solution with a concentration of 0.05% to 0.10% for 4 to 8 hours after germination.

[0018] By adopting the above technical solution, the two-step efficient identification of haploids was achieved by combining coleoptile color labeling for initial selection with flow cytometry ploidy detection. Combined with the colchicine root soaking method for chromosome doubling, the selected hybrid offspring can obtain homozygous double haploid systems in a short period of time, providing a material basis with consistent genetic background for subsequent combining ability determination.

[0019] Preferably, in step S5, the test species belongs to a different heterotic group than the DH line to be tested, and the general combining ability of the test species has been confirmed by trials in no less than 3 locations and no less than 2 planting years. The test species has appeared as a parent at least twice in the varieties approved in the target ecological zone. Each DH line to be tested is simultaneously testcrossed with 3 test species.

[0020] By adopting the above technical solution, the test species are selected from the backbone lines of different heterotic groups from the DH lines to be tested. The general combining ability of the test species has been verified by multiple locations and years of trials and has appeared as a parent in approved varieties, ensuring the representativeness and reliability of the combining ability test results. Each DH line to be tested is simultaneously testcrossed with 3 test species, and the accuracy of combining ability evaluation is improved through multiple comparisons.

[0021] Preferably, in step S6, the association analysis involves sorting all DH lines within the same batch from high to low molecular marker scores to obtain sequence A, and sorting them from high to low measured combining ability to obtain sequence B. The rank correlation coefficient between sequence A and sequence B is calculated. Based on the association analysis results, molecular markers that are positively correlated with the measured combining ability are given a higher weight in subsequent batch screenings, while molecular markers that are negatively correlated with the measured combining ability are given a lower weight or are used as negative selection markers.

[0022] By adopting the above technical solution, a quantitative correlation between molecular markers and coordination ability is established by sorting all DH series in the same batch according to molecular marker scores and measured values ​​of coordination ability and calculating the rank correlation coefficient. Based on the analysis results, the weight of molecular markers that are positively correlated with the measured values ​​of coordination ability is increased, while the weight of negatively correlated markers is decreased or they are used as negative selection markers. This allows the molecular marker screening strategy for subsequent batches to be continuously optimized based on the previous measured data, thereby gradually improving the screening accuracy of high coordination ability materials.

[0023] Preferably, in step S7, the comprehensive rating includes molecular marker score, combining ability score, quality trait score, and agronomic trait score, and the top 10% of DH lines in the comprehensive rating enter the hybridization stage. The quality trait score is calculated by weighting the measured values ​​of test weight, crude protein content, and crude fat content, and the agronomic trait score is calculated by weighting the measured values ​​of plant height, ear height, growth period, disease resistance, and lodging resistance indicators with equal weights.

[0024] By adopting the above technical solution, the evaluation of multi-resistant maize germplasm resources is comprehensively scored using four indicators: molecular marker scoring, combining ability scoring, quality trait scoring, and agronomic trait scoring, with different weights. This allows the evaluation to cover multiple dimensions, including gene level, combining ability level, quality level, and agronomic level. The top 10% of DH lines in the comprehensive score enter the hybridization stage, effectively reducing the scale of materials entering field testing while ensuring excellent comprehensive traits.

[0025] In summary, this application has the following beneficial effects: 1. Because this application adopts differentiated hybridization introduction based on target traits of different functional groups, superior genes from different sources are introduced into backbone lines under various adaptive schemes. Combined with the feedback iteration of molecular marker continuous screening and combining ability determination results of each generation, the screening strategy is continuously optimized. Therefore, the efficiency and accuracy of introducing superior genes of local varieties into backbone inbred lines are improved, and the problem that the efficiency and accuracy of introducing superior genes of local varieties into backbone inbred lines in existing maize germplasm breeding methods are difficult to meet the needs of creating multi-resistant germplasm resources is solved.

[0026] 2. This application achieves the targeted introduction and generational locking of superior genes from donors by designing hybridization introduction schemes based on the different target traits of different functional groups and combining them with continuous screening of molecular markers in each generation. On this basis, the results of combining ability determination and molecular marker detection are correlated to adjust the screening weights, so that the screening criteria can be dynamically optimized based on actual breeding data. This forms a closed loop from gene introduction to continuous improvement of screening strategies, which improves the efficiency of introducing superior genes from local varieties and the accuracy of screening materials with superior combining ability.

[0027] 3. This application eliminates the extra waiting time of propagation followed by test cross in traditional methods by simultaneously completing self-pollination and combining ability test cross in the same planting season when obtaining DH lines. At the same time, the test cross results are used to optimize the molecular marker screening strategy for subsequent batches, so that the two goals of shortening the breeding cycle and improving the screening accuracy can be achieved synergistically in the same technical operation, effectively improving the breeding efficiency of multi-resistant hybrids. Attached Figure Description

[0028] Figure 1 This is a flowchart of the method provided in this application; Figure 2 The figure shows the experimental results of the breeding cycle and DH line acquisition efficiency of this application; Figure 3 This is a graph showing the experimental results of the retention rate of the target gene and the accuracy of the combining ability screening in this application; Figure 4 This is a graph showing the experimental results of the yield of the variety in this application. Detailed Implementation

[0029] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0030] Unless otherwise specified, all experimental methods used below are conventional methods. All materials, reagents, methods, and instruments used, unless otherwise specified, are conventional materials, reagents, methods, and instruments in this field, which can be obtained commercially or prepared according to literature methods by those skilled in the art.

[0031] To better understand the above technical solutions, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments.

[0032] Please see the appendix Figure 1 The following is a further description with reference to the embodiments: Example 1: A method for innovation of multi-resistant maize germplasm resources and hybrid breeding, including the following steps: S1. Collect local variety resources, including Longshuiti Laohuang Corn, Jin Huanghou, Baima Ya, Xiaoli Hong, Baitou Shuang, Yeji Hong, Gui Zongxuan, etc.; plant each local variety according to a completely randomized block design, with 50 plants per material, standard row spacing of 60cm and plant spacing of 25cm, and conventional field management, recording agronomic traits during the growth period, including the total number of days of growth, plant height, and ear height. After harvest, select 10 representative ears from each material to examine ear length, number of rows per ear, and 100-kernel weight. After harvest, randomly sample 500g of kernels from each local variety and use a near-infrared spectroscopy analyzer to test quality traits, including test weight, crude protein content, and crude fat content; use artificial inoculation identification method to identify resistance to sheath blight, embedding infected toothpicks into the leaf sheath at the base of the stem at the corn jointing stage, one toothpick per plant, inoculating 30 plants per material, and investigating the disease level according to the 1-9 standard at maturity and calculating the disease index; Based on the identification results, and using the main superior traits of each variety as the classification criteria, Longshuiti Laohuang Corn and Yejihong were classified into the stress-resistant functional group; Jinhuanghou and Baimaya were classified into the quality-oriented functional group; Baitoushuang was classified into the weather-resistant functional group; and Xiaolihong and Guizongxuan were classified into the high-yield functional group.

[0033] S2, Three-stage hybridization of stress-resistant functional groups: First hybridization: Using Longshuiti old yellow corn as the female parent and the backbone line A of the same group as the male parent, hybridization was carried out to prepare 15 ears of hybrids. After mixed harvesting, F1 generation seeds were obtained. F1 generation seeds were planted in drought-stressed plots with soil moisture content controlled at 50% of field capacity, with a population size of 200 plants and no additional irrigation. At maturity, single plants that grew well under drought stress, had coordinated male and female ears, normal grain filling, and no obvious disease symptoms were selected, with the selection rate not exceeding 30% of the total population. Each selected single plant was bagged and self-pollinated, and harvested by division, with one ear selected from each plant. Second hybridization: The self-pollinated seeds of the selected single plants from the first hybridization were planted in rows in a plot under double stress: on the basis of drought stress, artificial inoculation with sheath blight was carried out at the jointing stage, with a population size of 100 plants. At the maturity stage, drought resistance, disease resistance and agronomic traits were comprehensively evaluated, and single plants with drought resistance, disease resistance and excellent agronomic traits were selected. The selection rate was no more than 30% of the total population. Each selected single plant was bagged and self-pollinated, and then harvested by division. One ear of fruit was selected from each plant. Third hybridization: The self-pollinated seeds of the selected single plants from the second hybridization were planted in conventional water and fertilizer management plots in rows of ears, with a population size of 100 plants. At maturity, single plants with excellent comprehensive traits were selected according to the breeding objectives. 18 excellent ears of fruit were selected from each row of ears, mixed, threshed and stored. Backbone lines from different sources but belonging to the same hybrid vigor group were used as male parents for each hybridization. Two hybridizations of the quality-type functional group: First hybridization: Using Golden Queen as the female parent and Q1, the backbone line of the same group, as the male parent, hybridization was carried out to prepare 15 hybrid combinations of ears. After mixed harvesting, F1 generation seeds were obtained. The F1 generation seeds were planted in conventionally managed plots with a population size of 200 plants. At maturity, preliminary selection was carried out according to the standards of excellent agronomic traits, with the selection rate not exceeding 30% of the total population. Each selected plant was bagged and self-pollinated, and then harvested by division. Second hybridization: Self-pollinated seeds from the selected plants in the first hybridization were planted in conventionally managed plots, with a population size of 100 plants per ear. Each plant was tagged at the tasseling and silking stage. After maturity, the plants were harvested separately and threshed. 100g of grains from each plant were thoroughly mixed and sampled. Near-infrared spectroscopy was used to analyze the test weight, crude protein content, and crude fat content. Plants were selected based on the following quality thresholds: test weight ≥720g / L, crude protein content ≥10%, and crude fat content ≥4%. Plants meeting all three criteria were selected, and 18 superior ears were retained per ear row. Three hybridizations of the weather-resistant functional group: First hybridization: Using Baitoushuang as the female parent and T1, the backbone line of the same group, as the male parent, hybridization was carried out to prepare 15 hybrid ears. After mixed harvesting, F1 generation seeds were obtained. F1 generation seeds were sown in the season when the highest daily temperature is ≥35℃, with a population size of 200 plants. During the pollen shedding and silking stage, the coordination of male and female ears was observed. Individual plants with large pollen shedding, smooth silking, and strong silk vigor were selected and tagged. The selection rate at maturity did not exceed 25% of the total population. Second hybridization: The self-pollinated seeds of the selected single plants from the first hybridization are planted in conventionally managed plots in rows, with a population size of 100 plants. At maturity, select single plants with moderate growth period, moderate plant height, and large fruit ears, and retain 18 fruit ears per row. Third hybridization: The seeds of the selected ears from the second hybridization are planted in conventionally managed plots according to the ear rows, with a population size of 100 plants. At maturity, single plants with excellent comprehensive traits are selected according to the breeding objectives. 18 excellent ears are selected from each ear row. The backbone lines from different sources but belonging to the same hybrid vigor group are used as male parents for each hybridization. Three hybridizations of the high-yielding functional group: The first to third hybridizations were all planted in conventionally managed plots with population sizes of 200, 100, and 100 plants respectively. After each hybridization, individual plants with outstanding ear traits were selected: ear length ≥20cm, ear diameter ≥4.5cm, number of ear rows ≥16, and 100-grain weight ≥30g. At least three of the four indicators must be met to be selected. 18 ears were selected from each ear row. Backbone lines from different sources but belonging to the same hybrid vigor group were used as male parents for each hybridization. S3. Screening for SSR molecular markers closely linked to the target trait. The marker combinations used for each functional group are as follows: 10 markers related to stress resistance; 7 markers related to quality; 5 markers related to weather resistance; and 8 markers related to high yield. Each marker is polymorphic between the donor local variety and the recipient backbone line, and its association is verified in control materials with known target traits.

[0034] After the first hybridization: Young leaves (approximately 2 cm²) were taken from selected individual plants, and genomic DNA was extracted using the CTAB method. Using the extracted DNA as a template, PCR amplification was performed using four molecular markers associated with the core target trait. The PCR reaction system (20 μL) contained: 50–100 ng DNA template, 0.4 μmol / L each of forward and reverse primers, 200 μmol / L each of dNTPs, 1 U of Taq DNA polymerase, and 1× PCR buffer (containing Mg²⁺). + 1.5 mmol / L); PCR amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, annealing (temperature set according to the Tm value of each primer) for 45 s, 72℃ extension for 60 s, for a total of 32 cycles; 72℃ final extension for 7 min; the amplification products were detected by 8% non-denaturing polyacrylamide gel electrophoresis, and the genotype of each single plant at each marker site was recorded. Single plants that did not carry any donor target gene markers were eliminated, and single plants carrying at least 2 target markers were retained for the second hybridization. After the second hybridization: DNA was extracted from the selected individual plants and molecular marker detection was performed. Extended marker combinations were used: 10 stress-resistant, 7 quality, 5 weather-resistant, and 8 high-yielding. Molecular marker fingerprints were established based on the genotype results of each individual plant at each marker locus. The population was sorted according to the number of target gene markers carried, and the top 30% of individual plants were retained for the third hybridization. After the third hybridization: DNA was extracted from the endosperm tissue of each seed from the selected ears, and a complete set of molecular markers was used for genotyping verification: 15 stress-resistant types, 10 quality types, 8 weather-resistant types, and 12 high-yielding types. Ears with a donor target gene marker loss rate exceeding 30% were eliminated. S4. Using the BC2F1 generation individuals that have passed the verification of S3 as the female parent and the haploid inducing line carrying the Navajo marker as the male parent for hybridization induction, the female parent is selected before sowing, and the male parent is sown in stages to ensure that the flowering period coincides with that of the female parent. During the pollination period, the female parent is manually bagged for isolation. Each female parent ear is pollinated by the male parent three times, once a day from 9 to 11 am for three consecutive days. After pollination, the ear is bagged again until the bracts of the ear turn yellow. After harvesting the ear, it is naturally air-dried. After air-drying and threshing, the kernels of each ear were initially selected based on the color of the coleoptile: kernels with no purple coleoptile were initially identified as haploid kernels, while kernels with purple coleoptiles were identified as diploid heterozygous kernels and were discarded. For the haploid kernels obtained in the initial selection, the embryo tissue was extracted and ploidy was rapidly detected using a flow cytometer: the embryo was placed in a sample tube, 500 μL of nuclear extraction buffer was added, the tissue was chopped with a sharp blade, allowed to stand for 5 min, filtered through a 30 μm filter, propidium iodide (final concentration 50 μg / mL) was added, and the sample was stained in the dark for 5 min before being detected by the flow cytometer, and the fluorescence intensity of the sample peak was recorded; using a known diploid maize embryo as a control, samples with a DNA content 0.5 times that of the control were identified as haploid and retained. Grains confirmed as haploids were germinated. When the radicle length reached 0.75 cm, chromosome doubling was performed using the root soaking method: the germinated seeds were immersed in a 0.08% colchicine solution containing 2% dimethyl sulfoxide for 6 hours at 20-25°C in the dark. The treated seeds were rinsed with running tap water for 30 minutes and then sown in greenhouse seedling trays with a substrate ratio of peat moss:vermiculite:perlite = 3:1:1. The plants obtained after doubling were cultivated in the greenhouse until maturity. Each plant was self-pollinated and bagged, and harvested individually. The doubling success was determined according to the following criteria: (1) normal plant morphology and complete development of male and female ears; (2) self-pollination and fruit formation with grains in the ears; (3) consistent phenotype of offspring plants and no segregation of traits. Materials that meet all three criteria are confirmed as DH lines and numbered and stored. S5. The selection of test species shall meet the following criteria: it shall not belong to the heterotic group of the DH line to be tested; the general combining ability of the test species shall be proven to be high through trials at no less than 3 locations and for no less than 2 planting years; the test species shall have appeared as a parent at no less than 2 times among the varieties approved in the target ecological zone; each DH line to be tested shall be testcrossed with 3 test species that meet the above criteria at the same time. The DH seeds obtained from S4 were planted in the isolation area in rows, with 20 plants per DH line. All plants were bagged before tasseling and silking, and self-pollinated. After maturity, the plants were harvested separately by line, and seeds of each line were retained for subsequent propagation. During the same planting season, another batch of DH seeds was planted in the testcross area according to the testcross scheme. Each DH line was hybridized with 3 test varieties. Using the DH line as the female parent and the test varieties as the male parent, artificial bagging and pollination were carried out. Each hybrid combination of DH line and each test variety was used to produce 5 ears of fruit. After harvesting, the ears were threshed and stored separately to obtain testcross varieties. After the self-pollinated ears of grain are harvested, 300g of mixed grain samples are taken from each DH line. The bulk density, crude protein content and crude fat content are detected by near-infrared spectroscopy. Each sample is measured three times and the average value is taken. At the same time, a natural disease survey of sheath blight is carried out in the field of DH line. The obtained test crosses were used to conduct yield comparison experiments in the next planting season. The experiment adopted a randomized complete block design with 3 replicates. The plot area was 12m², the planting density was 4800 plants / mu, and the local main cultivar was used as the control. After maturity, all plots were harvested and the yield was calculated. The average yield of each DH line and each test cross was used as the measured value of the general combining ability of the DH line. S6. Establish an individual file for each DH line, including genotype data of the DH line at each stage of molecular marker continuous screening in S3, DH induction status in S4, agronomic and quality trait identification data and measured values ​​of combining ability in S5. Within the same batch, sort all DH lines from high to low molecular marker scores to obtain sequence A, and sort them from high to low combined ability to obtain sequence B. Calculate the Spearman rank correlation coefficient between sequence A and sequence B. Based on the correlation analysis results, the weight of molecular markers that are positively correlated with the measured value of combining ability is increased in subsequent batch screenings, while the weight of molecular markers that are negatively correlated with the measured value of combining ability is reduced or used as negative selection markers. The adjusted marker weight scheme is recorded and archived to guide the molecular marker screening of the next batch of breeding materials. S7. All DH systems that have completed the combination ability test are comprehensively scored according to the following four indicators: Molecular marker score: The score is based on the percentage of target gene markers carried by each DH line out of the total number of target markers, with a maximum score of 100.

[0035] Combined succession score: Based on the highest average yield of all DH line testcrosses in this batch, the combined succession score of each DH line is calculated as (average yield of the DH line testcross / highest yield) × 100.

[0036] Quality trait scoring: Based on the measured values ​​of three indicators—bulk weight, crude protein content, and crude fat content—each indicator is assigned a value according to the following standards: ≥740g / L: 100 points; 720–740g / L: 80 points; 700–720g / L: 60 points; <700g / L: 40 points; Crude protein content: ≥12%: 100 points; 10%–12%: 80 points; 8%–10%: 60 points; <8%: 40 points; Crude fat content: ≥5%: 100 points; 4%–5%: 80 points; 3%–4%: 60 points; <3%: 40 points; The weighted average of the three scores (weight: bulk weight 40%, protein 30%, fat 30%) is used as the quality score. Agronomic trait scoring: The agronomic trait score is comprehensively evaluated based on five indicators: plant height, ear height, growth period, disease resistance, and lodging resistance. Each indicator is assigned a percentage score based on the measured value, and then the scores are calculated by weighting them equally. The four scores are calculated using the following weightings: molecular marker score (20%), measured combining ability score (40%), quality trait score (20%), and agronomic trait score (20%). The overall score is calculated as follows: Overall Score = Molecular Marker Score × 0.2 + Measured Combining Ability Score × 0.4 + Quality Trait Score × 0.2 + Agronomic Trait Score × 0.2. The top 10% of DH lines in the comprehensive score were rated as Grade A and entered the targeted hybridization stage. Grade A DH lines were targeted for hybridization according to the principle of complementary heterosis groups. The maternal parent group selected Grade A DH lines of the SS, Reid, or X groups, and the paternal parent group selected Grade A DH lines of the Huanggai, Lv, Lanca, tropical germplasm, P, or Iodent groups to prepare hybrid combinations. The prepared hybrid combinations were selected for multi-point yield comparison trials at 5 representative locations in the target ecological zone. The trials adopted a randomized complete block design with 3 replicates. The plot area was 15m², and the planting density was 4800 plants / mu. At the harvest, the yield, test weight, crude protein content, crude fat content, sheath blight incidence, and lodging rate were measured at each experimental site. The hybrid combination that performed stably at most experimental sites was selected, with a yield not less than 105% of the control variety, a test weight ≥720g / L, a crude protein content ≥10%, and a crude fat content ≥4%, thus completing the breeding process.

[0037] Example 2: This example differs from Example 1 above in that: In S1, 50 plants were planted in each material, with a standard row spacing of 60cm and a plant spacing of 25cm. Ten representative ears of each local variety were selected for ear trait assessment. 500g of grains were sampled from each local variety for quality testing. 30 plants were inoculated with sheath blight in each material.

[0038] In S2, for the stress-resistant functional group, the first hybridization involves creating 20 hybrid combinations for each ear of fruit. The F1 generation population size is 200 plants, with a selection rate not exceeding 30% of the total population. The second hybridization population size is 100 plants, with a selection rate not exceeding 30% of the total population. The third hybridization population size is 100 plants, with 20 superior ears selected from each row. For the quality-oriented functional group, the first hybridization involves creating 20 hybrid combinations for each ear of fruit. The F1 generation population size is 200 plants, with a selection rate not exceeding 30% of the total population. The second hybridization population size is 100 plants, with 20 superior ears selected from each row. The quality index thresholds are: test weight ≥ 720 g / L, crude protein content ≥ 10%, and crude fat content ≥ 4%. For the weather-resistant functional group, the first hybridization involves preparing 20 hybrid combinations and sowing them in seasons with a maximum daily temperature of ≥35℃. The population size is 200 plants, and the selection rate is no more than 25% of the total population. For the second hybridization, the population size is 100 plants, and 20 ears are selected from each row. For the third hybridization, the population size is 100 plants, and 20 superior ears are selected from each row. For the high-yielding functional group, the population sizes for the first to third hybridizations are 200 plants, 100 plants, and 100 plants, respectively. The selection criteria for ear traits are ear length ≥20cm, ear diameter ≥4.5cm, number of rows ≥16, and 100-kernel weight ≥30g. 20 ears are selected from each row.

[0039] In S3, there were 12 markers related to stress resistance, 8 markers related to quality, 6 markers related to weather resistance, and 10 markers related to high yield. After the first hybridization, 5 molecular markers were used for screening, with 35 PCR amplification cycles. After the second hybridization, an expanded marker combination was used: 12 markers for stress resistance, 8 for quality, 6 for weather resistance, and 10 for high yield. The top 30% of individual plants were retained. After the third hybridization, a complete set of molecular markers (20 for stress resistance, 12 for quality, 10 for weather resistance, and 15 for high yield) was used for validation, and ears of fruit with a donor target gene marker loss rate exceeding 30% were discarded.

[0040] In S4, each female ear was pollinated with male pollen 3 times, the colchicine solution concentration was 0.10%, the treatment time was 8 hours, and the plants after double treatment were identified according to the following standards: (1) the plant morphology was normal and the male and female ears were fully developed; (2) the plant was self-pollinated and had grains formed in the ear; (3) the offspring plants had the same phenotype and no trait segregation.

[0041] In S5, 20 plants were planted in each DH line. Five ears of fruit were produced by hybridizing each DH line with each test variety. 300g of mixed grain samples were taken from each DH line for quality testing. Each test variety was tested three times. The yield comparison experiment of the test cross was conducted using a randomized complete block design with three replicates. The plot area was 15m² and the planting density was 5000 plants / mu.

[0042] In S6, all DH lines within the same batch are sorted from high to low molecular marker scores to obtain sequence A, and sorted from high to low measured combining power to obtain sequence B. Spearman rank correlation coefficients are calculated, and molecular markers that are positively correlated with measured combining power are given higher weights, while molecular markers that are negatively correlated with measured combining power are given lower weights or are used as negative selection markers.

[0043] In S7, the quality trait scoring criteria are as follows: Test weight ≥740g / L: 100 points; 720-740g / L: 80 points; 700-720g / L: 60 points; <700g / L: 40 points; Crude protein content ≥12%: 100 points; 10%-12%: 80 points; 8%-10%: 60 points; <8%: 40 points; Crude fat content ≥5%: 100 points; 4%-5%: 80 points; 3%-4%: 60 points; <3%: 40 points; The weighting of the three items is: test weight 40%, protein 30%, fat 30%; Agronomic traits are scored based on plant height, ear height, growth period, etc. Disease resistance and lodging resistance were calculated using a weighted average of five indicators; the comprehensive score was calculated as follows: molecular marker score × 0.2 + combined ability score × 0.4 + quality trait score × 0.2 + agronomic trait score × 0.2. The top 10% of DH lines in terms of comprehensive score entered the hybridization stage. Five representative locations were selected for the multi-site yield comparison trial. The trial adopted a randomized complete block design with three replicates. The plot area was 20 m², the planting density was 5000 plants / mu, and the screening criteria were: yield not less than 105% of the control variety, test weight ≥ 720 g / L, crude protein content ≥ 10%, and crude fat content ≥ 4%.

[0044] Example 3: This example differs from Example 1 above in that: In S1, 50 plants were planted in each material, with a standard row spacing of 60cm and a plant spacing of 25cm. Ten representative ears of each local variety were selected for ear trait assessment. 500g of grains were sampled from each local variety for quality testing. 30 plants were inoculated with sheath blight in each material.

[0045] In S2, for the stress-resistant functional group, the first hybridization involves creating 15 hybrid combinations for each ear; the F1 generation population size is 200 plants, with a selection rate not exceeding 30% of the total population; the second hybridization population size is 100 plants, with a selection rate not exceeding 30% of the total population; the third hybridization population size is 100 plants, with 15 superior ears selected from each row. For the quality-oriented functional group, the first hybridization involves creating 15 hybrid combinations for each ear; the F1 generation population size is 200 plants, with a selection rate not exceeding 30% of the total population; the second hybridization population size is 100 plants, with 15 superior ears selected from each row. Quality index thresholds: test weight ≥720g / L, crude protein content ≥10%, crude fat content ≥4%. For the weather-resistant functional group, the first hybridization involves preparing 15 hybrid combinations and sowing them in seasons with a maximum daily temperature of ≥35℃. The population size is 200 plants, and the selection rate is no more than 25% of the total population size. For the second hybridization, the population size is 100 plants, and 15 ears are selected from each row. For the third hybridization, the population size is 100 plants, and 15 superior ears are selected from each row. For the high-yielding functional group, the population sizes for the first to third hybridizations are 200 plants, 100 plants, and 100 plants, respectively. The selection criteria for ear traits are ear length ≥20cm, ear diameter ≥4.5cm, number of rows ≥16, and 100-kernel weight ≥30g. 15 ears are selected from each row.

[0046] In S3, there were 8 markers related to stress resistance, 5 markers related to quality, 4 markers related to weather resistance, and 6 markers related to high yield. After the first hybridization, 3 molecular markers were used for screening, and the number of PCR amplification cycles was 30. After the second hybridization, an expanded marker combination was used: 8 for stress resistance, 5 for quality, 4 for weather resistance, and 6 for high yield, and the top 30% of individual plants were retained. After the third hybridization, a complete set of molecular markers was used: 12 for stress resistance, 8 for quality, 6 for weather resistance, and 10 for high yield, for verification, and ears of fruit with a donor target gene marker loss rate of more than 30% were eliminated.

[0047] In S4, each female ear was pollinated with male pollen 3 times, the colchicine solution concentration was 0.05%, the treatment time was 4 hours, and the plants after double treatment were identified according to the following standards: (1) the plant morphology was normal and the male and female ears were fully developed; (2) the plant was self-pollinated and had grains formed in the ears; (3) the offspring plants had the same phenotype and no trait segregation.

[0048] In S5, 20 plants were planted in each DH line, and 5 ears of fruit were prepared by hybridization of each DH line with each test species. 300g of mixed grain samples were taken from each DH line for quality testing. Each test was repeated 3 times. The yield comparison experiment of the test cross was conducted using a randomized complete block design with 3 replicates. The plot area was 10m², and the planting density was 4500 plants / mu.

[0049] In S6, all DH lines within the same batch are sorted from high to low molecular marker scores to obtain sequence A, and sorted from high to low measured combining power to obtain sequence B. Spearman rank correlation coefficients are calculated, and molecular markers that are positively correlated with measured combining power are given higher weights, while molecular markers that are negatively correlated with measured combining power are given lower weights or are used as negative selection markers.

[0050] In S7, the quality trait scoring criteria are as follows: ≥740g / L test weight: 100 points; 720–740g / L test weight: 80 points; 700–720g / L test weight: 60 points; <700g / L test weight: 40 points; ≥12% crude protein content: 100 points; 10%–12% crude protein content: 80 points; 8%–10% crude protein content: 60 points; <8% crude protein content: 40 points; ≥5% crude fat content: 100 points; 4%–5% crude fat content: 80 points; 3%–4% crude fat content: 60 points; <3% crude fat content: 40%. The weighting of the three scores is: test weight 40%, protein 30%, and fat 30%. Agronomic trait scores were calculated using a weighted average of five indicators: plant height, ear height, growth period, disease resistance, and lodging resistance. The comprehensive score was calculated as follows: Molecular marker score × 0.2 + Measured combining ability score × 0.4 + Quality trait score × 0.2 + Agronomic trait score × 0.2. The top 10% of DH lines in terms of comprehensive score entered the hybridization stage. For the multi-site yield comparison trial, five representative locations were selected. The trial adopted a randomized complete block design with three replicates. The plot area was 15 m², and the planting density was 4500 plants / mu. The screening criteria were: yield not less than 105% of the control variety, test weight ≥ 720 g / L, crude protein content ≥ 10%, and crude fat content ≥ 4%.

[0051] Comparative Example 1: The difference between this comparative example and Example 1 is that: The data feedback and label weight iteration in step S6 are not performed. That is, after step S5 is completed, the comprehensive evaluation in step S7 is performed directly. The molecular label screening weight remains unchanged throughout the process, and the rest is the same as in Example 1.

[0052] Comparative Example 2: This comparative example differs from Example 1 above in that: In step S2, all functional groups adopt the same hybridization introduction scheme and no differentiated design is carried out - all adopt the 3-hybridization scheme and no targeted selection pressure is applied (the stress-resistant functional group is not subjected to the dual stress of drought and sheath blight, the weather-resistant functional group is not sown in the high-temperature season, the quality-type functional group is not subject to quality tracking, and the high-yield functional group is not subject to selection for the enhancement of ear traits), and the rest is the same as in Example 1.

[0053] Comparative Example 3: This comparative example differs from Example 1 above in that: In step S3, molecular marker screening is performed only after the last hybridization. Molecular marker detection and screening are not performed after the first two hybridizations. That is, selection is based solely on phenotypic traits after the first and second hybridizations. Genotype verification is performed only after the third hybridization using a full set of molecular markers. The rest is the same as in Example 1.

[0054] Comparative Example 4: This comparative example differs from Example 1 above in that: In step S3, the number of molecular markers used after each hybridization is the same (15 each), and the stage-specific marker combination strategy is not adopted (i.e., a small number of core markers are not used in the early stage and the number of markers is gradually increased in the later stage). The rest is the same as in Example 1.

[0055] Comparative Example 5: This comparative example differs from Example 1 above in that: In step S5, after obtaining the DH line, it is first self-pollinated for two seasons to obtain sufficient seeds, and then a combining ability test cross is performed. Instead of simultaneously performing self-pollination and test cross mating in the first planting season after obtaining the DH line, the rest is the same as in Example 1.

[0056] Performance testing: The breeding effects of Examples 1-3 and Comparative Examples 1-5 were tested as follows: Breeding cycle: Record the number of planting seasons required from the start of parental hybridization to obtaining a hybrid that can enter multi-location trials, in seasons, with each season counted as one growth cycle; Target gene retention rate: Molecular marker detection was used, and the percentage of selected ears that still carried the donor target gene marker after the third hybridization in step S3 was used to measure the retention rate. The detection markers were the core markers in the complete set of molecular markers in S3, and the five markers with the highest correlation with the target trait were selected from each functional group. Combination ability screening accuracy: The percentage of DH series whose measured combination ability ranking is indeed in the top 10% of the batch among the top 10% of the A-grade DH series in step S7, out of the total number of A-grade DH series. DH line acquisition efficiency: The number of DH lines that ultimately meet the identification criteria out of every 100 BC2F1 individuals screened by molecular markers in step S4; Variety yield: The average yield (kg / mu) of the hybrids obtained in the final screening of each example and comparative example in the multi-point test was standardized based on the average yield of the control variety at each test point; The test results are shown in Table 1.

[0057] Table 1: Breeding effect test data of each embodiment and comparative example

[0058] In conjunction with Examples 1 to 3 and Comparative Examples 1 to 5, and in conjunction with Table 1 and Figures 1 to 4 It can be seen that through the coordinated efforts of each step, a complete breeding process is formed. Step S1 provides the basis for donor classification for differentiated hybridization. Step S2 implements targeted introduction based on the classification results. Step S3 performs quality control on the hybrid generation. Step S4 achieves rapid purification. Step S5 simultaneously completes seed retention and combining ability determination. Step S6 feeds the determination results back to the screening strategy to form a closed-loop optimization. Step S7 completes comprehensive evaluation and produces hybrids.

[0059] In conjunction with Example 1 and Comparative Example 1, and in conjunction with Table 1, Figure 3 , Figure 4 It can be seen that the data feedback and marker weight iteration in step S6 can improve the accuracy of combining ability screening. This step achieves closed-loop optimization of the screening strategy by performing correlation analysis between the measured combining ability value and the molecular marker genotype data and adjusting the weight of the molecular marker in subsequent batch screenings based on the analysis results. Without this step, the molecular marker weight cannot be dynamically adjusted according to the measured combining ability results, which leads to the inability to improve the accuracy of subsequent batch screenings and ultimately affects the yield level of hybrids.

[0060] In conjunction with Example 1 and Comparative Example 2, and in conjunction with Table 1 and Figure 1 , Figure 3 It can be seen that the differential hybridization introduction in step S2 is the key to ensuring the effective introduction of target traits of different functional groups. This scheme designs different hybridization times and selection pressures for different trait characteristics of stress-resistant, quality-oriented, weather-resistant, and high-yielding functional groups. Without differential design, the superior genes of various functional groups cannot be specifically preserved in the homogenization scheme, resulting in a reduced retention rate of target genes and ultimately affecting the yield performance of hybrids.

[0061] In conjunction with Example 1 and Comparative Example 3, and in conjunction with Table 1 and Figure 1 , Figure 3 It can be seen that the continuous screening of molecular markers after each hybridization in step S3 can ensure that the target gene is continuously retained in each generation. Without this continuous screening, individuals that do not carry the target gene in the first two generations are not eliminated in time, resulting in the dilution of the target gene frequency in the population generation by generation. Through continuous screening in each generation, it is ensured that the population entering the next round of hybridization always maintains a high target gene frequency.

[0062] In conjunction with Example 1 and Comparative Example 4, and in conjunction with Table 1 and Figure 1 , Figure 2 , Figure 3It can be seen that the stage-specific marker combination strategy in step S3 can balance the early screening accuracy and the protection of genetic diversity. This strategy uses a small number of core markers to protect genetic diversity in the early stage and a full set of markers to ensure screening accuracy in the later stage. When the same number of markers are used in each generation, the elimination criteria may be too strict due to too many markers in the early stage, which may lead to the wrong elimination of individuals carrying the target gene. In the later stage, the complete preservation status of the target gene cannot be fully verified due to insufficient markers.

[0063] In conjunction with Example 1 and Comparative Example 5, and in conjunction with Table 1 and Figure 1 , Figure 2 It can be seen that in step S5, performing self-pollination and combining ability testcross simultaneously during the first planting season of the DH line can shorten the breeding cycle. This step allows testcross mating and DH line propagation to be completed in parallel within the same growth cycle. When the testcross is delayed, the breeding cycle is significantly extended, while the yield level of the variety remains basically unchanged, indicating that the main contribution of this step is to shorten the breeding cycle rather than increase the yield.

[0064] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for innovation of multi-resistant maize germplasm resources and breeding of hybrid varieties, characterized in that, Includes the following steps: S1. Conduct a preliminary assessment of local varietal resources and classify local varieties into different functional groups based on the results of the preliminary assessment; S2. Based on the target traits of different functional groups, design differentiated hybridization introduction schemes to introduce the superior genes of local varieties into the backbone inbred lines for hybridization. S3. In the hybridization population, genotyping is performed using molecular markers, and individuals carrying the target gene marker are selected based on the test results; S4. Haploid induction is performed on the selected hybrid offspring, and chromosome doubling is performed on the obtained haploids to obtain a homozygous double haploid system, namely the DH line. S5. Simultaneously perform self-pollination and test crosses with the test species to determine the combining ability of each DH line. S6. Perform correlation analysis between the combination ability determination results and the molecular marker detection results, and adjust the screening weight of the molecular markers based on the correlation analysis results; S7. The DH lines are comprehensively rated based on molecular marker scores, combining ability scores, quality trait scores, and agronomic trait scores. The DH lines with the highest scores are then used for hybridization to complete the breeding process.

2. The method for innovation of multi-resistant maize germplasm resources and breeding of hybrids according to claim 1, characterized in that: In step S1, the functional groups include stress-resistant functional groups, quality-oriented functional groups, weather-resistant functional groups, and high-yield functional groups; the local varieties include Longshuiti Laohuang Corn, Jin Huanghou, Baima Ya, Xiaoli Hong, Baitoushuang, Yeji Hong, and Guizongxuan.

3. The method for innovation of multi-resistant maize germplasm resources and breeding of hybrids according to claim 1, characterized in that: In step S2, the differentiated hybridization introduction scheme is as follows: the stress-resistant functional group and the weather-resistant functional group adopt a 3-hybridization scheme, the quality functional group adopts a 2-hybridization scheme or a 3-hybridization scheme, and the high-yielding functional group adopts a 3-hybridization scheme. In each hybridization, 15 to 20 excellent ears are selected from the offspring population according to the breeding target trait. In each hybridization, the backbone lines from different sources but belonging to the same hybrid vigor group are used as the male parent.

4. The method for innovation of multi-resistant maize germplasm resources and breeding of hybrids according to claim 3, characterized in that: In the three-hybridization scheme, the number of female parent plants planted in the first hybridization shall not be less than 200, and the number of female parent plants planted in the second and third hybridization shall not be less than 100 each; the first hybridization of the weather-resistant functional group shall be sown in the season when the daily maximum temperature is ≥35℃.

5. The method for innovation of multi-resistant maize germplasm resources and breeding of hybrids according to claim 3, characterized in that: In the two-hybridization scheme of the quality-type functional group, after the second hybridization, the grains of the selected single plants are tested for quality traits, and the ears of single plants with a test weight ≥720g / L, crude protein content ≥10% and crude fat content ≥4% are retained.

6. The method for innovation of multi-resistant maize germplasm resources and breeding of hybrids according to claim 1, characterized in that: In step S3, during the screening, 3 to 5 molecular markers are used for screening after the first hybridization, 8 to 12 molecular markers are used for screening after the second hybridization, and 12 to 20 molecular markers are used for verification after the third hybridization. In each generation of screening, individuals carrying the top 30% of the target markers are retained, and after the third hybridization, ears of fruit with a target gene marker loss rate of more than 30% are eliminated.

7. The method for innovation of multi-resistant maize germplasm resources and breeding of hybrids according to claim 1, characterized in that: In step S4, the identification of haploids includes initial selection using coleoptile color marking, followed by flow cytometry analysis of the ploidy of the haploid kernels obtained from the initial selection, with known diploid maize embryos as a control. Samples with DNA content 0.45 to 0.55 times that of the control are identified as haploids. Chromosome doubling is performed using a root immersion method, in which haploid kernels are germinated and then immersed in a 0.05% to 0.10% colchicine solution for 4 to 8 hours.

8. The method for innovation of multi-resistant maize germplasm resources and breeding of hybrids according to claim 1, characterized in that: In step S5, the test species belongs to a different heterotic group than the DH line to be tested, and the general combining ability of the test species has been confirmed by trials in no less than 3 locations and no less than 2 planting years. The test species has appeared as a parent at least twice in the varieties approved in the target ecological zone. Each DH line to be tested is simultaneously testcrossed with 3 test species.

9. The method for innovation of multi-resistant maize germplasm resources and breeding of hybrids according to claim 1, characterized in that: In step S6, the association analysis involves sorting all DH lines within the same batch from high to low molecular marker scores to obtain sequence A, and sorting them from high to low measured combining ability to obtain sequence B. The rank correlation coefficient between sequence A and sequence B is calculated. Based on the association analysis results, molecular markers that are positively correlated with measured combining ability are given a higher weight in subsequent batch screenings, while molecular markers that are negatively correlated with measured combining ability are given a lower weight or are used as negative selection markers.

10. The method for innovation of multi-resistant maize germplasm resources and breeding of hybrids according to claim 1, characterized in that: In step S7, the comprehensive rating includes molecular marker score, combining ability score, quality trait score, and agronomic trait score. The top 10% of DH lines in the comprehensive rating enter the hybridization stage. The quality trait score is calculated by weighting the measured values ​​of test weight, crude protein content, and crude fat content. The agronomic trait score is calculated by weighting the measured values ​​of plant height, ear height, growth period, disease resistance, and lodging resistance indicators with equal weights.

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