Molecular marker for auxiliary screening of nitrogen efficient corn germplasm and application of molecular marker

Abstract

The invention discloses molecular markers for auxiliary screening of nitrogen efficient corn germplasm and application of the molecular markers, and relates to the technical field of biology, two molecular markers, namely chr6150749382CG and chr6150749401AG, highly associated with chlorate sensitivity are developed, the two markers can effectively distinguish chlorate sensitivity levels of a corn material, haplotype CA / CA shows high sensitivity, GG / GG shows low sensitivity, and the two markers can effectively distinguish chlorate sensitivity levels of the corn material. Therefore, the molecular marker combination can be used as a basis for preliminarily identifying the sensitivity (nitrogen absorption and utilization efficiency) of the chlorate of the corn, has high efficiency and applicability such as simplicity in operation, low cost and short time consumption, and has important values for digging nitrogen-efficient corn germplasm, improving nitrogen utilization efficiency, reducing nitrogen fertilizer application and promoting sustainable development of agriculture.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to molecular markers for assisting in screening nitrogen-efficient corn germplasm and applications thereof. Background Art

[0002] Corn (Zea mays L.), an important food, feed, and industrial crop, relies heavily on nitrogen fertilizer for yield increases. However, the gap between excessive nitrogen fertilizer application and corn's limited nitrogen utilization efficiency leads to significant nitrogen loss, causing environmental pollution and the need for fertilizer-tolerant crop varieties, hindering the sustainable development of agriculture. Breeding nitrogen-efficient corn varieties has become an urgent need for the industry.

[0003] Chlorate is a nitrate analogue that is toxic to plants. Plants absorb chlorate and nitrate through the same mechanism; the more sensitive a plant is to chlorate, the more efficient its nitrogen absorption is. Chlorate toxicity experiments offer the advantages of low cost, short turnaround times, and high phenotypic visualization, and have been successfully applied in studies of the genetics of nitrogen efficiency in rice. However, this technology has not been fully exploited in the analysis of the genetic basis of nitrogen efficiency in maize and in molecular breeding. There is an urgent need to establish an efficient breeding system by identifying key gene loci.

[0004] Traditional nitrogen-efficient corn breeding relies on field nitrogen stress phenotypic screening, which has significant limitations: (1) Difficulty in phenotypic identification: Nitrogen efficiency is regulated by multiple components such as root architecture and nitrogen transporter activity. Field phenotypes are easily affected by external environmental factors such as soil and climate, with low reproducibility, which can easily mask the true genetic effect; (2) Low selection efficiency: Constructing a low-nitrogen environment requires a lot of manpower and material resources, and the screening cycle is generally 3–5 years per generation, and it is difficult to distinguish the interaction effect between genotype and environment; (3) Slow genetic improvement: Nitrogen efficiency is essentially a complex quantitative trait with a complex regulatory network involving multiple physiological stages such as nitrogen absorption, transport, assimilation, and reuse. A single phenotype is difficult to fully reflect the gene control effect, and conventional hybrid breeding is difficult to efficiently aggregate dominant alleles. With the development of molecular biotechnology, marker-assisted selection (MAS) has gradually become an important supplementary means of high-efficiency breeding. However, the molecular markers currently used in nitrogen efficiency-related research are still mainly SSR and InDel, which have problems such as low detection efficiency, insufficient sensitivity, and poor automation level, which limit their promotion and application in large-scale germplasm resource screening and application.

[0005] KASP (Kompetitive Allele-Specific PCR) is a high-throughput genotyping technology based on SNP (single nucleotide polymorphism) differences. It has significant advantages such as high sensitivity, low cost, and simple operation. Based on dual-color fluorescent probes and universal primers, this technology can achieve closed-type, high-throughput automated genotyping on a standard real-time fluorescence PCR instrument. A single reaction can complete the genotyping of thousands of samples. Compared with traditional markers, KASP technology has the following significant features: (1) The detection throughput is increased by a hundred times, enabling batch detection of tens of thousands of samples, greatly improving screening efficiency, and supporting genome-wide association studies (GWAS) and high-throughput screening of breeding populations; (2) No electrophoresis or gel staining is required, and closed-tube detection is used to avoid contamination, with a genotyping accuracy of over 99%; (3) The detection cost per sample unit is low, and the reagent cost is reduced by more than 60%, significantly reducing breeding costs and facilitating its widespread application in commercial breeding.

[0006] Currently, KASP technology has shown promising application prospects in the research and molecular breeding of important agronomic traits in multiple crops. For example, Cao et al. (2022) precisely mapped genes associated with stalk rot resistance in maize and developed five KASP markers closely associated with resistance. These markers have been successfully used in breeding populations to assist in the aggregation of disease-resistant genes, improving the efficiency of breeding resistant lines. In maize, although studies have identified multiple QTLs and candidate genes closely related to nitrogen efficiency, dedicated KASP markers for nitrogen efficiency are still very limited, hindering the rapid integration and utilization of excellent genes. By deeply exploring genome-wide SNP variation under nitrogen response conditions and combining nitrogen efficiency phenotypic data with GWAS analysis and candidate gene screening, a precisely mapped KASP marker system can be constructed, providing strong support for the rapid screening and aggregation breeding of subsequent nitrogen-efficient materials, breaking through the genetic bottleneck of traditional breeding.

[0007] In summary, the construction of KASP molecular markers suitable for maize nitrogen-efficient traits can not only effectively make up for the shortcomings of traditional molecular markers in this field, but also promote the development of maize breeding from empirical selection to high-precision and high-efficiency selection, and provide key technical guarantees for the realization of green agricultural production increases, nitrogen fertilizer reduction and sustainable development goals. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide molecular markers for assisting in screening nitrogen-efficient corn germplasm and applications thereof.

[0009] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0010] A method for screening or assisting in screening nitrogen-efficient corn germplasm, comprising the following steps: detecting whether the genotype of the corn to be tested is CA / CA or GG / GG, wherein the nitrogen utilization efficiency of the corn with the genotype CA / CA is greater than the nitrogen utilization efficiency of the corn with the genotype GG / GG;

[0011] The corn of the CA / CA genotype is a corn of the CA / CA homozygous genotype based on the C51G SNP site and the A51G SNP site;

[0012] The corn of the genotype GG / GG is a corn of homozygous GG / GG genotype based on the C51G SNP site and the A51G SNP site;

[0013] The C51G SNP site is the 150749382nd SNP on chromosome 6 of the maize Zm-B73-REFERENCE-GRAMENE-4.0 reference genome, corresponding to the 51st nucleotide from the 5' end of SEQ ID NO: 1;

[0014] The A51G SNP site is the 150749401st SNP on chromosome 6 of the maize Zm-B73-REFERENCE-GRAMENE-4.0 reference genome, corresponding to the 51st nucleotide from the 5' end of SEQ ID NO: 2.

[0015] Further preferably, the step of detecting whether the genotype of the corn to be tested is genotype CA / CA or genotype GG / GG is as follows:

[0016] (a1) using the genomic DNA of the corn to be tested as a template, performing PCR amplification using two sets of primer combinations to obtain a PCR amplification product;

[0017] The first primer combination consists of an upstream primer F1 shown in SEQ ID NO: 3, an upstream primer F2 shown in SEQ ID NO: 4, and a downstream primer R1 shown in SEQ ID NO: 5;

[0018] The second primer combination consists of upstream primer F3 shown in SEQ ID NO: 6, upstream primer F4 shown in SEQ ID NO: 7, and downstream primer R2 shown in SEQ ID NO: 8;

[0019] (a2) After completing step (a1), the fluorescence signal of the PCR amplification product is detected by an instrument, and the genotype of the corn to be tested is obtained according to the color of the fluorescence signal. If the fluorescence of the amplification product is consistent with the fluorescence of the fluorescent group labeled with primers F1 and F3, and exhibits blue fluorescence, then the corn sample to be tested is of CA / CA genotype; if the fluorescence of the amplification product is consistent with the fluorescence of the fluorescent group labeled with primers F2 and F4, and exhibits yellow fluorescence, then the corn sample to be tested is of GG / GG genotype.

[0020] Further preferably, the step of detecting whether the genotype of the corn to be tested is genotype CA / CA or genotype GG / GG is as follows:

[0021] (b1) using the genomic DNA of the corn to be tested as a template, performing PCR amplification using two sets of primer combinations to obtain a PCR amplification product;

[0022] The first primer combination consists of an upstream primer F1 shown in SEQ ID NO: 3, an upstream primer F2 shown in SEQ ID NO: 4, and a downstream primer R1 shown in SEQ ID NO: 5;

[0023] The second primer combination consists of upstream primer F3 shown in SEQ ID NO: 6, upstream primer F4 shown in SEQ ID NO: 7, and downstream primer R2 shown in SEQ ID NO: 8;

[0024] (b2) taking the PCR amplification product obtained in step (b1) and sequencing it;

[0025] (b3) Obtaining the genotype of the corn to be tested based on the sequencing results obtained in step (b2).

[0026] Use of a SNP site, the SNP site is located at chr6_150749382 on chromosome 6 of the maize Zm-B73-REFERENCE-GRAMENE-4.0 reference genome, corresponding to base 51 of the nucleotide sequence shown in SEQ ID NO: 1. When the site is homozygous for CC, the corresponding genotype is Q1.382.a; when it is homozygous for GG, the corresponding genotype is Q1.382.b;

[0027] The use is to screen or assist in screening nitrogen-efficient corn, and the different nitrogen utilization efficiencies are: corn carrying the Q1.382.a genotype is higher or has the potential to be higher than corn carrying the Q1.382.b genotype.

[0028] Use of a SNP site, the SNP site is located at chr6_150749401 on chromosome 6 of the maize Zm-B73-REFERENCE-GRAMENE-4.0 reference genome, corresponding to base 51 of the nucleotide sequence shown in SEQ ID NO: 2. When the site is homozygous for AA, the corresponding genotype is Q2.401.a; when it is homozygous for GG, the corresponding genotype is Q2.401.b;

[0029] The use is to screen or assist in screening nitrogen-efficient corn, and the different nitrogen utilization efficiencies are: corn carrying the Q2.401.a genotype is higher or has a potential to be higher than corn carrying the Q2.401.b genotype.

[0030] A kit for identifying or assisting in identifying the nitrogen utilization efficiency of corn, comprising a primer combination for detecting whether the genotype of the corn to be tested is genotype CC or genotype GG;

[0031] The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 3, an upstream primer F2 shown in SEQ ID NO: 4, and a downstream primer R1 shown in SEQ ID NO: 5;

[0032] The genotype CC is based on the C51G SNP site, which is a CC homozygous type;

[0033] The genotype GG is based on the genotype of the C51G SNP site being a GG homozygous type;

[0034] The C51G SNP site is the 150749382nd SNP on chromosome 6 of the maize Zm-B73-REFERENCE-GRAMENE-4.0 reference genome, corresponding to the 51st nucleotide from the 5' end of SEQ ID NO: 1.

[0035] A kit for identifying or assisting in identifying the nitrogen utilization efficiency of corn, comprising a primer combination for detecting whether the genotype of the corn to be tested is genotype AA or genotype GG;

[0036] The primer combination consists of an upstream primer F3 shown in SEQ ID NO: 6, an upstream primer F4 shown in SEQ ID NO: 7, and a downstream primer R2 shown in SEQ ID NO: 8;

[0037] The genotype AA is based on the genotype of the A51G SNP site being AA homozygous;

[0038] The genotype GG is based on the genotype of the A51G SNP site being a GG homozygous type;

[0039] The A51G SNP site is the 150749401st SNP on chromosome 6 of the maize Zm-B73-REFERENCE-GRAMENE-4.0 reference genome, corresponding to the 51st nucleotide from the 5' end of SEQ ID NO: 2.

[0040] The molecular marker shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0041] Application of the above kit or the above molecular marker in identifying or assisting in identifying the nitrogen utilization efficiency of corn.

[0042] The use of a primer combination in the directed breeding or assisted directed breeding of nitrogen-efficient corn lines, the primer combination comprising any one or a combination of the following:

[0043] A first primer combination consisting of an upstream primer F1 represented by SEQ ID NO: 3, an upstream primer F2 represented by SEQ ID NO: 4, and a downstream primer R1 represented by SEQ ID NO: 5;

[0044] The second primer combination consists of the upstream primer F3 shown in SEQ ID NO: 6, the upstream primer F4 shown in SEQ ID NO: 7 and the downstream primer R2 shown in SEQ ID NO: 8.

[0045] The beneficial effects of the above technical solution are as follows: By testing chlorate sensitivity in an AMP-associated population of 386 genetically diverse maize inbred lines and combining it with a high-density genetic linkage map for genome-wide association analysis, the present invention successfully located a significant association locus on maize chromosome 6. Further linkage disequilibrium (LD) analysis within ±100 kb of this locus led to the development of two molecular markers highly associated with chlorate sensitivity: chr6_150749382_C_G and chr6_150749401_A_G. These two markers can effectively differentiate the chlorate sensitivity levels of maize materials: haplotypes CA / CA indicate high sensitivity, while GG / GG indicate low sensitivity. Identification results in the AMP population and populations from different breeding eras in China and the United States demonstrate the good stability and applicability of this marker combination. Therefore, the molecular marker combination provided by the present invention can be used as a rapid, simple and low-cost preliminary screening tool for chlorate sensitivity, providing technical support for the discovery and breeding of efficient nitrogen utilization corn germplasm resources, and has important practical application value in reducing nitrogen fertilizer usage, improving nitrogen fertilizer utilization efficiency and promoting sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1This is a diagram of the chlorate hydroponic experimental process of 386 AMP groups in the present invention.

[0047] Figure 2 This is a diagram of the process of obtaining the chlorate hydroponic phenotype of 386 AMP populations in the present invention.

[0048] Figure 3 The two materials with the largest sensitivity difference calculated by aboveground fresh weight from the 386 AMP populations screened in the chlorate experiment of the present invention are: the material with the highest chlorate sensitivity: GEMS29, and the material with the lowest chlorate sensitivity: GEMS30. The scale is 3 cm.

[0049] Figure 4 Normal distribution plot of phenotypic data of chlorate sensitivity calculated for aboveground fresh weight of 386 AMP populations.

[0050] Figure 5 Manhattan plot of GWAS results calculated using a mixed linear model (MLM) for aboveground chlorate sensitivity of 386 AMP populations.

[0051] Figure 6 Schematic diagram of the association analysis results between maize germplasm grouped with genotype Q1.382.a and genotype Q1.382.b and the mean chlorate sensitivity at the seedling stage in 386 AMP maize inbred lines under the hydroponic chlorate experiment ("*" indicates P < 0.05, i.e., the difference reaches a significant level; "**" and "***" indicate P < 0.01 and P < 0.001, respectively, i.e., the difference reaches an extremely significant level).

[0052] Figure 7 The KASP typing results of chr6_150749382 locus of 96 maize materials of Sino-American age are shown.

[0053] Figure 8 Schematic diagram of the correlation analysis results between the mean nitrogen concentrations in the stems and plants of 96 Sino-American maize accessions sown in Shuihuang Village, Kaifeng City, Henan Province in 2023 and the KASP-typed maize germplasm ("*" indicates P < 0.05, i.e., the difference reaches a significant level; "**" and "***" indicate P < 0.01 and P < 0.001, respectively, i.e., the difference reaches an extremely significant level). DETAILED DESCRIPTION

[0054] The following examples illustrate the present invention in detail. The various raw materials and equipment used in the present invention are conventional commercial products and can be directly obtained through commercial purchase. The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0055] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of described features, integers, steps, operations, elements and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or collections thereof.

[0056] It will also be understood that the term "and / or" used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

[0057] References to "one embodiment" or "some embodiments" in this specification mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in one or more embodiments of the present application. Thus, phrases such as "in one embodiment," "in some embodiments," "in other embodiments," and "in other embodiments" appearing in various places in this specification do not necessarily refer to the same embodiment, but rather mean "one or more but not all embodiments," unless otherwise specifically emphasized. The terms "including," "comprising," "having," and variations thereof all mean "including but not limited to," unless otherwise specifically emphasized.

[0058] In addition, in the description of the present application specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the descriptions and cannot be understood as indicating or implying relative importance.

[0059] The following is a clear and complete description of the technical solution of the present invention in conjunction with the specific embodiments of the present invention.

[0060] However, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0061] Example 1, plant materials

[0062] The maize AMP population used in this study is the Association Mapping Panel (AMP), a collection of 527 maize inbred lines with tropical, subtropical, and temperate genetic backgrounds, exhibiting extensive phenotypic diversity and complex genetic relationships. This population has been systematically described by Yang et al. (2011) (Yang, X. et al., Mol Breeding, 28, 511–526). A total of 386 accessions were randomly selected from this population for subsequent experiments.

[0063] Example 2. Experimental design and phenotype acquisition

[0064] like Figure 1 As shown, 386 selected AMP corn materials were planted in the indoor greenhouse conditions of Huazhong Agricultural University for hydroponics. Ten plants were set for each material and divided into the control (CK) group and the treatment (T) group, with 5 biological replicates in each group. The culture conditions were a photoperiod of 14 hours of light / 10 hours of darkness, and the day and night temperatures were 30°C / 28°C, respectively. The hydroponic solution used was a modified Kimura nutrient solution (the formula of the basic nitrogen-free complete hydroponic nutrient solution is detailed in Table 1), and all reagents were sourced from Shanghai Shanghai-type analytical reagents. The treatments were as follows: control group (CK): basic nitrogen-free nutrient solution + 0.5mM KNO3 (increased to 2.5mMKNO3 after the start of treatment); treatment group (T): basic nitrogen-free nutrient solution + 0.5mM KNO3 (0.5mM KClO3 + 2.0mM KNO3 after the start of treatment). As shown Figure 2-3 As shown, after chlorate treatment for 5 days, the fresh weight of the aboveground part of the plants was measured using a Deant JY20002 electronic balance (accuracy 0.01g). Phenotypic data were processed using R-4.3.2 software, and outliers were removed using the IQR method. The mean of each trait was calculated, and chlorate sensitivity (Chlorate-sensitivity) was calculated according to the following formula:

[0065]

[0066] The normal distribution of phenotypic data of chlorate sensitivity calculated from aboveground fresh weight is shown in Figure 4 .

[0067] Table 1 Basic nitrogen-free complete hydroponic nutrient solution formula

[0068]

[0069]

[0070] Example 3. Genome-wide association study (GWAS)

[0071] SNP quality control was performed on 386 maize inbred lines using Plink software. The filtering criteria were missing rate (NA) > 20% and minimum allele frequency (MAF) < 0.05. Approximately 13 million high-quality SNP markers were finally obtained. Genome-wide association analysis was performed using GAPIT software. A mixed linear model (MLM) was used to analyze chlorate sensitivity calculated from aboveground fresh weight. A Manhattan plot was drawn (see Figure 5). -log10(p)≥6.36 was set as the significance threshold of the association analysis. It was found that there was a relatively continuous interval site with -log10(p)≥6.36 in the Chr6:150705382-150848566 interval of the tested maize chromosome 6. The ANOVA function of R-4.3.2 software was used for variance analysis, and P<0.05 was used as the significance level to judge the significance of the phenotypic differences between different haplotype materials (see Figure 6 ).

[0072] Table 2 Statistical analysis of the relationship between allelic variation types of chr6_150749382 and chr6_150749401 in AMP maize and chlorate sensitivity

[0073]

[0074] Example 4. Development and application of KASP molecular markers

[0075] Based on an analysis of significant chlorate sensitivity-associated loci located on maize chromosome 6 (Chr6:150705382–150848566), the present invention ultimately identified two key SNPs: chr6_150749382_C_G and chr6_150749401_A_G. Based on the principles of KASP technology, the inventors combined base variation information at these two loci and, after extensive experimental optimization, successfully designed and synthesized KASP molecular markers for amplification. Two pairs of KASP primer combinations were designed, each consisting of two upstream allele-specific primers and a universal downstream primer, for efficient and specific identification of samples with different genotypes. All primers were synthesized by Shanghai Sangon Biotech Co., Ltd.; the specific nucleotide sequences are detailed in Table 3.

[0076] Different allele types are divided into the following two steps: PCR amplification and genotyping. The methods used in the examples are conventional methods unless otherwise specified.

[0077] (1) PCR amplification system and procedure: Genomic DNA from maize leaves was extracted using the CTAB method and fully dissolved in 90 μl of ddH2O before subsequent processing. DNA quality was assessed by electrophoresis on a 1% agarose gel, requiring clear bands, no obvious impurities or degradation, and an A260 / A280 ratio of approximately 1.8 as measured by UV spectrophotometry. After determining the DNA concentration, all materials were uniformly diluted to 30–40 ng / μl and used as templates in the PCR reactions.

[0078] Preparation of KASP labeled primer working solution: Dilute the F1 and F2 primers in Table 3 to 36 μmol / l and the R primer to 90 μmol / l. Then, aspirate 12 μl of each of the two upstream primers and 30 μl of the downstream primer of the primer combination, add sterile ultrapure water to 100 μl, mix well, and use as KASP primer MIX working solution. Store at -20°C in the dark until used.

[0079] Table 3 KASP marker primer sequences for allelic variations of chr6_150749382 and chr6_150749401 in the Sino-American breeding population

[0080]

[0081] The PCR amplification system was as follows: template DNA 4 μl, primer MIX working solution 0.14 μl, 2×KASP Master Mix (LGC, product number: KBS-1050-102) 5 μl, and ddH2O 1 μl.

[0082] The PCR reaction procedure is as follows: pre-denaturation at 94°C for 15 min; 10 cycles of denaturation at 94°C for 20 s and annealing for 20 s (the first annealing temperature is 61°C, and the temperature is lowered by 0.6°C each cycle); 34 cycles of denaturation at 94°C for 20 s and annealing at 55°C for 1 min; and storage at 12°C. It is recommended to use a white opaque PCR plate during the experiment to ensure the fluorescence signal detection effect.

[0083] (2) Genotyping: After PCR amplification is completed, the fluorescence signal of the amplified product is detected using a BIO-RAD CFX series fluorescence quantitative PCR instrument, and the fluorescence signal is converted into analyzable numerical data. The obtained fluorescence data is graphically displayed using the "ggplot2" package in the R-4.3.2 software, so as to accurately determine the sample genotype. The genotype is determined based on the type of fluorescence signal presented by the amplified product: if the fluorescence signal of the amplified product is consistent with the fluorescence of the fluorescent group labeled with primers F1 and F3, and shows blue fluorescence, then the corn sample to be tested is determined to be a nitrogen-efficient material; if the fluorescence of the amplified product is consistent with the fluorescence of the fluorescent group labeled with primers F2 and F4, and shows yellow fluorescence, then the corn sample to be tested is determined to be a nitrogen-inefficient material; in the above system, samples that do not show obvious fluorescence signals may be of deletion or heterozygous type. ( Figure 7 ).

[0084] Example 5: Detection of Chlorate Sensitivity in Chinese and American Breeding Maize Populations Using KASP Markers

[0085] A total of 320 maize inbred lines from Sino-American breeding years were selected and sown in Shuihuang Village, Kaifeng City, Henan Province (geographic coordinates: 34°29′N, 114°11′E) in May 2023. A randomized block design was used, with 13 plants planted per accession, a row spacing of 0.5 m, and a plant spacing of 0.2 m.

[0086] 96 samples were randomly selected from 320 samples for genotyping analysis. The genomic DNA of the samples was extracted using the CTAB method and amplified and SNP typed using the KASP primers provided by the present invention. The KASP reaction system and procedure are shown in Example 4. The genotyping results are shown in Figure 4. Figure 7 As shown: at the chr6_150749382 locus, a total of 43 materials were detected to be CC homozygous alleles, and 53 materials were detected to be GG homozygous alleles; at the chr6_150749401 locus, a total of 42 materials were detected to be AA homozygous alleles, 53 were detected to be GG homozygous alleles, and another 1 material was detected to be heterozygous.

[0087] Example 6. Correlation analysis between four maize genotypes (genotype Q1.382.a and genotype Q1.381.b) and field nitrogen content:

[0088] To further verify the application value of KASP markers in the identification of nitrogen efficiency traits, this example measured the nitrogen accumulation of 96 maize population materials from Chinese and American breeding years after harvest, and conducted association analysis based on the KASP genotyping results.

[0089] After field plants matured and were harvested, leaves and stems from each sample were processed separately. Samples were first dried at 80°C to constant weight and then thoroughly ground into a powder using a grinder. For each sample, 90–110 mg of powder was weighed and wrapped in tin foil. Nitrogen concentration was determined using the Dumas combustion method using a rapid nitrogen analyzer (Rapid N Exceed, Elementar, Langenselbold, Germany). Before each measurement, the instrument was calibrated using three standard asparagine samples as reference standards. After calibration, the labels and weights of all standard and test samples were entered into the Rapid N Exceed software (v.1.4.0) and placed in the designated order on the sample tray for automated measurement. After measurement, the raw data for all test samples were corrected using the nitrogen concentrations of the standard samples. The corrected data were exported to Excel for subsequent analysis. The average nitrogen concentration of the plant was calculated by averaging the nitrogen concentrations of the stems and leaves.

[0090] The field stem nitrogen concentration and average nitrogen concentration of 96 maize inbred lines from China and the United States breeding era and the corresponding detected SNP genotypes are shown in Table 4.

[0091] Table 4 Detection results of chr6_150749382 and chr6_150749401 markers of maize germplasm Kasp and nitrogen concentration data of maize tissues in Shuihuang Village, Kaifeng City

[0092]

[0093]

[0094]

[0095] Table 5 Statistical analysis of the relationship between allelic variation types of chr6_150749382 and chr6_150749401 and SNC (%) in maize from the Sino-American era

[0096]

[0097] Table 6 Statistical analysis of the relationship between allelic variant types of chr6_150749382 and chr6_150749401 and ANC (%) in maize from the Sino-American era

[0098]

[0099] Note: Statistical analysis was performed using a two-tailed t-test; * indicates significant differences, and ** indicates extremely significant differences.

[0100] The results are as follows Figure 8 , it can be seen that the stem nitrogen concentration and plant average nitrogen concentration of the KASP molecular marker site chr6_150749382 - C:C site are significantly higher than those of the G:G site, while the stem nitrogen concentration of the chr6_150749401 site A:A site is significantly higher than that of the G:G site, but there is no significant difference in the average plant nitrogen concentration. This result shows that the contribution of the chr6_150749382 site to corn nitrogen is greater than that of the chr6_150749401 site. (As shown in Table 5, Table 6, Figure 8 ).

[0101] As can be seen from the above examples, the present invention collected 386 genetically diverse AMP-associated maize inbred lines, cultivated them hydroponically with chlorate, measured their phenotypes, and sequenced them. Combined with a high-density genetic linkage map, the study pinpointed a locus on chromosome 6 significantly associated with maize chlorate sensitivity. Linkage disequilibrium analysis of SNPs within a ±100 kb region led to the development of molecular markers, chr6_150749382_C_G and chr6_150749401_A_G, that are tightly linked to the chlorate sensitivity trait. These two markers accurately differentiate maize AMP populations and populations from Sino-American breeding years into those with high chlorate sensitivity (haplotype CA / CA) and those with low chlorate sensitivity (haplotype GG / GG). Therefore, this molecular marker combination can serve as a basis for preliminary identification of maize chlorate sensitivity (nitrogen absorption and utilization efficiency), offering high efficiency and applicability, including simplicity, low cost, and time efficiency. This combination is of great value in identifying nitrogen-efficient maize germplasm, improving nitrogen use efficiency, reducing nitrogen fertilizer application, and promoting sustainable agricultural development.

[0102] The above results show that the KASP marker provided by the present invention has good discriminatory ability and applicability in maize age groups. The marker can be effectively used for the rapid identification of maize nitrogen-efficient genotypes, providing a reliable tool support for breeding screening and precise material selection.

[0103] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these examples without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0104] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant description of other embodiments.

[0105] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention, and should all be included in the scope of protection of the present invention.

Claims

1. A method for screening or assisting in screening nitrogen-efficient corn germplasm, characterized in that: The method comprises the following steps: detecting whether the genotype of the corn to be tested is CA / CA or GG / GG, wherein the nitrogen utilization efficiency of the corn with the genotype CA / CA is greater than the nitrogen utilization efficiency of the corn with the genotype GG / GG; The corn of the CA / CA genotype is a corn of the CA / CA homozygous genotype based on the C51G SNP site and the A51G SNP site; The corn of the genotype GG / GG is a corn of homozygous GG / GG genotype based on the C51G SNP site and the A51G SNP site; The C51G SNP site is based on the maize Zm-B73-REFERENCE-GRAMENE- 4.0 SNP 150749382 on chromosome 6 of the reference genome, corresponding to nucleotide 51 from the 5' end of SEQ ID NO: 1; The A51G SNP site is based on the maize Zm-B73-REFERENCE-GRAMENE- The 150749401st SNP on chromosome 6 of the 4.0 reference genome corresponds to the 51st nucleotide from the 5' end of SEQ ID NO:

2.

2. The method according to claim 1, characterized in that The steps of detecting whether the genotype of the corn to be tested is genotype CA / CA or genotype GG / GG are as follows: (a1) using the genomic DNA of the corn to be tested as a template, performing PCR amplification using two sets of primer combinations to obtain a PCR amplification product; The first primer combination consists of an upstream primer F1 shown in SEQ ID NO: 3, an upstream primer F2 shown in SEQ ID NO: 4, and a downstream primer R1 shown in SEQ ID NO: 5; The second primer combination consists of upstream primer F3 shown in SEQ ID NO: 6, upstream primer F4 shown in SEQ ID NO: 7, and downstream primer R2 shown in SEQ ID NO: 8; (a2) After completing step (a1), the fluorescence signal of the PCR amplification product is detected by an instrument, and the genotype of the corn to be tested is obtained according to the color of the fluorescence signal. If the fluorescence of the amplification product is consistent with the fluorescence of the fluorescent group labeled with primers F1 and F3, and exhibits blue fluorescence, then the corn sample to be tested is of CA / CA genotype; if the fluorescence of the amplification product is consistent with the fluorescence of the fluorescent group labeled with primers F2 and F4, and exhibits yellow fluorescence, then the corn sample to be tested is of GG / GG genotype.

3. The method according to claim 1, characterized in that The steps of detecting whether the genotype of the corn to be tested is genotype CA / CA or genotype GG / GG are as follows: (b1) using the genomic DNA of the corn to be tested as a template, performing PCR amplification using two sets of primer combinations to obtain a PCR amplification product; The first primer combination consists of an upstream primer F1 shown in SEQ ID NO: 3, an upstream primer F2 shown in SEQ ID NO: 4, and a downstream primer R1 shown in SEQ ID NO: 5; The second primer combination consists of upstream primer F3 shown in SEQ ID NO: 6, upstream primer F4 shown in SEQ ID NO: 7, and downstream primer R2 shown in SEQ ID NO: 8; (b2) taking the PCR amplification product obtained in step (b1) and sequencing it; (b3) Obtaining the genotype of the corn to be tested based on the sequencing results obtained in step (b2).

4. The use of SNP sites is characterized by: The SNP site is located at Zm-B73-REFERENCE-GRAMENE- chr6_150749382 on chromosome 6 of the 4.0 reference genome corresponds to base 51 of the nucleotide sequence shown in SEQ ID NO:

1. When the site is homozygous for CC, the corresponding genotype is Q1.382.a; when it is homozygous for GG, the corresponding genotype is Q1.382.b; The use is to screen or assist in screening nitrogen-efficient corn, and the different nitrogen utilization efficiencies are: corn carrying the Q1.382.a genotype is higher or has the potential to be higher than corn carrying the Q1.382.b genotype.

5. The use of SNP sites, characterized in that, The SNP site is located at Zm-B73-REFERENCE-GRAMENE- chr6_150749401 on chromosome 6 of the 4.0 reference genome corresponds to base 51 of the nucleotide sequence shown in SEQ ID NO:

2. When the site is homozygous for AA, the corresponding genotype is Q2.401.a; when it is homozygous for GG, the corresponding genotype is Q2.401.b; The use is to screen or assist in screening nitrogen-efficient corn, and the different nitrogen utilization efficiencies are: corn carrying the Q2.401.a genotype is higher or has a potential to be higher than corn carrying the Q2.401.b genotype.

6. A kit for identifying or assisting in identifying the nitrogen utilization efficiency of corn, characterized in that: The invention comprises a primer combination for detecting whether the genotype of the corn to be tested is genotype CC or genotype GG; The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 3, an upstream primer F2 shown in SEQ ID NO: 4, and a downstream primer R1 shown in SEQ ID NO: 5; The genotype CC is based on the C51G SNP site, which is a CC homozygous type; The genotype GG is based on the genotype of the C51G SNP site being a GG homozygous type; The C51G SNP site is based on the maize Zm-B73-REFERENCE-GRAMENE- The 150749382nd SNP on chromosome 6 of the 4.0 reference genome corresponds to the 51st nucleotide from the 5' end of SEQ ID NO:

1.

7. A kit for identifying or assisting in identifying the nitrogen utilization efficiency of corn, characterized in that: The invention comprises a primer combination for detecting whether the genotype of the corn to be tested is genotype AA or genotype GG; The primer combination consists of an upstream primer F3 shown in SEQ ID NO: 6, an upstream primer F4 shown in SEQ ID NO: 7, and a downstream primer R2 shown in SEQ ID NO: 8; The genotype AA is based on the genotype of the A51G SNP site being AA homozygous; The genotype GG is based on the genotype of the A51G SNP site being a GG homozygous type; The A51G SNP site is based on the maize Zm-B73-REFERENCE-GRAMENE- The 150749401st SNP on chromosome 6 of the 4.0 reference genome corresponds to the 51st nucleotide from the 5' end of SEQ ID NO:

2.

8. The molecular marker represented by SEQ ID NO: 1 or SEQ ID NO:

2.

9. Use of the kit according to claim 6 or 7 or the molecular marker according to claim 8 in identifying or assisting in identifying the nitrogen utilization efficiency of corn.

10. Use of a primer combination in the directed breeding or assisted directed breeding of nitrogen-efficient maize lines, the primer combination comprising any one or a combination of the following: A first primer combination consisting of an upstream primer F1 represented by SEQ ID NO: 3, an upstream primer F2 represented by SEQ ID NO: 4, and a downstream primer R1 represented by SEQ ID NO: 5; The second primer combination consists of the upstream primer F3 shown in SEQ ID NO: 6, the upstream primer F4 shown in SEQ ID NO: 7, and the downstream primer R2 shown in SEQ ID NO: 8.

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