An SV molecular marker related to nitrogen use efficiency in maize and its application

Abstract

The present invention discloses an SV molecular marker associated with the nitrogen use efficiency trait of maize and its application. The SV molecular marker is located on bases 6722060 to 6724624 of chromosome 5 of the maize B73 genome with the genome version number GRAMENE-4.0, and its sequence is shown in SEQ ID NO. 3. Based on the SV molecular marker, the present invention designs corresponding primers and a detection kit to achieve the prediction and screening of the maize nitrogen use efficiency trait, providing a scientific basis for breeding maize inbred lines with different nitrogen use efficiency types.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an SV molecular marker related to the nitrogen utilization efficiency trait of corn and an application thereof. Background Art

[0002] As my country's largest grain crop, maize (Zea mays L.) faces the key challenges of ensuring stable and high yields in the field of scientific breeding. Nitrogen use efficiency plays an extremely important role in the many agronomic traits of maize. In the maize production process, maize with low nitrogen use efficiency has difficulty absorbing excessive nitrogen fertilizers, which not only wastes nitrogen fertilizer resources, but also causes many environmental problems, such as soil and water pollution, and increased greenhouse gas emissions. The high nitrogen use efficiency of maize is closely linked to the realization of green and efficient production. Improving the nitrogen use efficiency of maize is of great significance. It can not only reduce the amount of nitrogen fertilizer applied in agricultural production, thereby reducing environmental pollution and greenhouse gas emissions, but also effectively increase crop yields, providing a solid guarantee for food security.

[0003] Currently, researchers have achieved many results in the localization of genetic loci for nitrogen utilization efficiency in maize. For example, Zhang et al. successfully located 73 genetic loci related to the activity of nitrogen metabolism enzymes using the offspring separation populations of B73 and Mo17, 10 of which belong to key enzyme sites for nitrogen metabolism. Hirel et al. conducted genetic analysis of physiological traits such as yield and nitrogen metabolism, and identified a series of important genetic loci including glutamine synthetase (GS1). However, existing technologies often use field phenotyping methods to mark maize nitrogen utilization traits. This method has obvious disadvantages and requires a large amount of manpower and material resources.

[0004] Genomic structural variation (SV) refers to changes in the length of sequences and positional relationships in the genome. It covers a variety of types, including long sequence insertions or deletions (Big Indels) longer than 50bp, tandem repeats, chromosomal inversions, translocations within or between chromosomes, copy number variations (CNVs), and more complex mosaic variations. Compared with other types of variation, SVs differ significantly in length, origin, function, and impact. SVs can detect structural variations of thousands of bases in the genome, and their markers exhibit co-dominant genetic characteristics, with good repeatability and stability.

[0005] In view of the above situation, how to effectively use molecular marker technology to predict the nitrogen utilization efficiency of corn and cultivate corn with high nitrogen utilization efficiency has become a technical problem that needs to be urgently solved in this field. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an SV molecular marker related to the nitrogen utilization efficiency trait of corn and an application thereof.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0008] The invention discloses an SV molecular marker related to the nitrogen use efficiency trait of maize. The SV molecular marker is located on bases 6722060 to 6724624 of chromosome 5 of the maize B73 genome with the genome version number GRAMENE-4.0, and its sequence is shown in SEQ ID NO.3.

[0009] A reagent for specifically detecting SV molecular markers located on bases 6722060 to 6724624 of chromosome 5 of the maize B73 genome with the genome version number GRAMENE-4.0.

[0010] A further optimized reagent for specifically detecting the SV molecular marker located at bases 6722060 to 6724624 of chromosome 5 of the maize B73 genome with the genome version number GRAMENE-4.0 is a primer pair for specifically amplifying the SV molecular marker.

[0011] Further optimized, the primer pair includes a forward primer ASD-F and a reverse primer ASD-R;

[0012] The nucleotide sequence of the forward primer ASD-F is shown in SEQ ID NO.1;

[0013] The nucleotide sequence of the reverse primer ASD-R is shown in SEQ ID NO.2.

[0014] A detection kit comprising the primer pair according to claim 3 or 4 and a PCR amplification reagent.

[0015] Application of SV molecular marker located at bases 6722060 to 6724624 of chromosome 5 of maize B73 genome with genome version number GRAMENE-4.0 in breeding of maize nitrogen use efficiency.

[0016] Application of SV molecular marker located at bases 6722060 to 6724624 of chromosome 5 of maize B73 genome with genome version number GRAMENE-4.0 in the identification of nitrogen use efficiency of maize.

[0017] Use of the reagent for specifically detecting the SV molecular marker according to any one of claims 2 to 4 in identifying and / or screening corn with different nitrogen utilization efficiencies.

[0018] Application of an SV molecular marker located on bases 6722060 to 6724624 of chromosome 5 of the maize B73 genome with the genome version number GRAMENE-4.0 or a detection kit containing the primer pair and PCR amplification reagent according to claim 3 or 4 in constructing a maize molecular ID card.

[0019] A method for identifying the nitrogen use efficiency trait of corn comprises the following steps:

[0020] Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using the primer pair described in claim 3 or 4 to obtain a PCR amplification product; performing gel electrophoresis on the PCR amplification product and making a judgment;

[0021] When the PCR amplification product shows a band at a length of 429 bp, the sample to be tested is a high nitrogen utilization type; when the PCR amplification product shows a band at a length of 3232 bp, the sample to be tested is a homozygous low nitrogen utilization efficiency type; when the PCR amplification product shows a band at a length of 429 bp and 3232 bp respectively, the sample to be tested is a heterozygous type.

[0022] The beneficial effects of the above technical solution are as follows: the present invention provides an SV molecular marker related to the nitrogen utilization efficiency trait of corn, the SV molecular marker is a large fragment insertion occurring at bases 6722060 to 6724624 on chromosome 5 of the corn B73 genome, and the nucleotide sequence of the large fragment is shown in SEQ ID NO.3; the version of the corn B73 genome is GRAMENE-4.0, and the SV molecular marker is significantly correlated with the nitrogen utilization trait of corn; in addition, the present invention designs corresponding primers and detection kits based on the SV molecular marker, thereby realizing the prediction and screening of the nitrogen utilization efficiency trait of corn, and providing a scientific basis for breeding corn inbred lines with different nitrogen utilization efficiency types. At the same time, during the detection process, there is no need to consider the growth period and tissue type of corn, nor is there any need to conduct field phenotypic investigations. The genotype information of the sample can be accurately and quickly obtained, which is beneficial to accelerate the germplasm innovation of high nitrogen utilization efficiency corn and improve breeding efficiency.

[0023] Based on the above technical advantages, the present invention also provides a method for identifying the nitrogen use efficiency trait in maize, comprising the following steps: using genomic DNA from a sample to be tested as a template, performing PCR amplification using the primers described in the above technical solution to obtain a PCR amplification product; and subjecting the PCR amplification product to gel electrophoresis and making an assessment. Experiments have demonstrated that the SV marker provided by the present invention and the primers designed based on the marker can accurately detect the nitrogen use efficiency trait in maize and effectively distinguish between homozygous heterozygous genotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the results of the genome-wide association analysis of nitrogen use efficiency of 421 maize diversity populations in Example 1, wherein: A is a Manhattan plot of the genome-wide association analysis of phenotypic values ​​of the 421 maize diversity populations under low nitrogen conditions; B is a Manhattan plot of the genome-wide association analysis of the ratio of phenotypic numbers of the 421 maize diversity populations under high nitrogen and low nitrogen conditions; C is a Manhattan plot of the genome-wide association analysis based on the expression levels of related genes of the 421 maize diversity populations;

[0025] Figure 2 Schematic diagram of the sequencing comparison results of the SV molecular marker intervals of corn materials with different nitrogen utilization efficiencies in Example 1;

[0026] Figure 3 This is a schematic diagram of the relative expression results of the primer set in Example 2 in materials with different nitrogen utilization efficiencies;

[0027] Figure 4 This is a schematic diagram of the molecular detection results of the primer set in Example 3 in materials with different nitrogen utilization efficiencies;

[0028] Figure 5 This is a bar graph showing the results of the nitrogen utilization efficiency survey of different maize inbred line materials based on molecular detection typing in Example 4;

[0029] Figure 6 This is a schematic diagram of the association analysis results between the status of gene polymorphism sites in natural populations and nitrogen utilization efficiency in Example 5. DETAILED DESCRIPTION

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. 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.

[0036] The research team of the present invention has provided an SV molecular marker associated with the nitrogen use efficiency trait of maize. The SV molecular marker is a large fragment inserted at bases 6722060 to 6724624 on chromosome 5 of the maize B73 genome. The nucleotide sequence of the large fragment is shown in SEQ ID NO. 3. The version of the maize B73 genome is GRAMENE-4.0; the download website of the maize B73 genome is https: / / download.maizegdb.org / Zm-B73-REFERENCE-GRAMENE-4.0 / , the genome database is named: MAIZEGDB; the maize B73 genome is publicly available in DOI: 10.1038 / nature22971.

[0037] Example 1: Acquisition of SV molecular markers related to maize nitrogen use efficiency traits

[0038] (1) Planting of diverse maize population materials, investigation of nitrogen use efficiency phenotypes and genome-wide association analysis:

[0039] In 2023, 421 diverse populations were planted in experimental fields in Xiangyang, Hubei Province. Nitrogen use efficiency (NUE) was measured in July and September, resulting in data on three NUE phenotypes. The 421 diverse populations and the selected maize materials with extreme NUE phenotypes were selected from the State Key Laboratory of Crop Genetic Improvement at Huazhong Agricultural University or from publicly available resequenced maize cultivars (see http: / / maizego.org / Resources.html and https: / / download.maizegdb.org for details).

[0040] Based on the 421 maize inbred lines whose genomes have been resequenced by the State Key Laboratory of Crop Genetic Improvement at Huazhong Agricultural University, we downloaded the resequencing data and combined them with 2 years of phenotypic data to conduct genome-wide association analysis. The results are shown in the attached figure. Figure 1 It can be seen that the nitrogen use efficiency data of the maize diversity population are all located at extremely significant sites in the same region of chromosome 5 throughout the genome.

[0041] Sequencing comparison results of extreme nitrogen utilization efficiency materials in the SV molecular marker interval:

[0042] The publicly available NAM inbred line and Chinese Founder inbred line resequencing data (data from Maizegdb, https: / / download.maizegdb.org) were used to compare the 6717165 to 6728461 base sequence interval of chromosome 5. The results are shown in the Appendix. Figure 2 , where the black marks indicate large insertions and deletions. We selected Zheng 58 and Chang 7-2, parents of the widely grown commercial hybrid Zhengdan 968, Jing 92 and Jing 724, parents of Jingke 968, a temperate material Ye 478, and a tropical material CML103. Figure 2Sequencing results revealed that the high-NUE materials (Jing 92, Chang 7-2, and CML103) contained an approximately 2.5 kb insertion compared to the low-NUE materials (Jing 724, Zheng 58, and Ye 478). This 2.5 kb fragment was designated as an SV marker; this variation was a large insertion-deletion variation within the SV marker. This SV marker is directly associated with the NUE trait in maize; plants with this insertion are likely to exhibit low NUE, while plants without the 2.5 kb insertion are likely to exhibit high NUE.

[0043] Example 2: Determining the accuracy of the effect of SV molecular markers on maize nitrogen use efficiency traits

[0044] To further confirm the accuracy of the SV molecular marker's effect on maize nitrogen use efficiency in Example 1, the physical location of the SV molecular marker was determined based on the maize B73 genome, GRAMENE-4.0, as the nucleotide sequence represented by bases 6722060 to 6724624 on maize chromosome 5. This sequence is located in an intron of the ASD1 gene, which is an amino acid metabolism gene shared across multiple species. The extreme materials CIMBL144, CY72, CML323, CIMBL16, TY8, 5213, GEMS27 and GEMS2 with low nitrogen use efficiency and the extreme materials D047, GEMS12, CIMBL95, H21, CIMBL152, CIMBL51 and GEMS35 with high nitrogen use efficiency were selected and planted. When the extreme materials reached the V7 stage under high nitrogen conditions (5 g urea per pot) and low nitrogen conditions (0 g urea per pot), the first fully expanded leaf from the top was taken and the total RNA of the different extreme materials was extracted using the kit method (RNA extraction kit purchased from Nanjing Novozymes Biotechnology Co., Ltd.). The expression level of ASD1 was detected by qRT-PCR. The results are shown in the attached figure. Figure 3 .

[0045] By the attached Figure 3 As can be seen, the expression level of the ASD1 gene T001 transcript in the high nitrogen use efficiency variety (ASD1) is 2.87 times higher than that in the low nitrogen use efficiency variety (ASD1-TE) under low nitrogen conditions and 4.37 times higher than that in the low nitrogen use efficiency variety (ASD1-TE) under high nitrogen conditions. This shows that the approximately 2.5 kb indel variant in Example 1 regulates the nitrogen use efficiency trait in maize by regulating its expression. Therefore, this large indel variant of approximately 2.5 kb is a key SV molecular marker affecting the nitrogen use efficiency trait in maize.

[0046] Example 3: PCR amplification and electrophoresis detection

[0047] Based on the SV molecular marker (about 2.5 kb) in Example 1, a specific primer set for identifying the nitrogen use efficiency trait of maize was designed. The specific primer set includes a forward primer ASD-F (SEQ ID NO. 1) and a reverse primer ASD-R (SEQ ID NO. 2);

[0048] The forward primer ASD-F and the reverse primer ASD-R can specifically amplify fragments of different nitrogen utilization efficiency genotypes with SV molecular marker insertions. The fragment size of 3232bp is a low nitrogen utilization efficiency genotype, and the corresponding genotype is Class II; the fragment size of 429bp is a high nitrogen utilization efficiency genotype, and the corresponding genotype is Class I.

[0049] Verify the accuracy of the primer set in step (1):

[0050] The extreme materials GEMS27 and GEMS2 with low nitrogen utilization efficiency and the extreme materials GEMS12, CIMBL95, CIMBL152 and CIMBL51 with high nitrogen utilization efficiency were used as the test materials, and the steps were as follows:

[0051] A. The CTAB method was used to extract maize genomic DNA from different extreme materials;

[0052] B. PCR amplification: Using the maize genomic DNA extracted in step A as a template, and using the primer set (SEQ ID NO.1 to SEQ ID NO.2) in step (1), PCR amplification was performed to obtain a PCR amplification product;

[0053] The PCR amplification system in 20 μL was as follows: 1 μL 30-150 ng / μL DNA template, 10 μL 2× TaqPlus MasterMix (Dye Plus), 1 μL ASD-F (10 μM), 1 μL ASD-R (10 μM), and 7 μL ddH2O.

[0054] The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; 34 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 210 s; extension at 72°C for 5 min, and storage at 10°C for 2 min. 2× Taq Plus Master Mix was purchased from Nanjing Novozymes Biotech Co., Ltd.

[0055] PCR agarose gel electrophoresis detection: Use 1% agarose gel to detect the PCR amplification product in step B. The results are shown in Figure 4 (exist Figure 4, the bars from left to right represent the results of the blank control, the results of the GEMS27 material, the results of the GEMS2 material, the results of the GEMS12 material, the results of the CIMBL95 material, the results of the CIMBL152 material, and the results of the CIMBL51 material).

[0056] Depend on Figure 4 As can be seen, after electrophoresis, the PCR amplification product of the low nitrogen utilization material only has a single band at 3232 bp, while the PCR amplification product of the extremely high nitrogen utilization efficiency material has a single band at 429 bp. This shows that the primer sets provided by the present invention, as shown in SEQ ID NO. 1 to SEQ ID NO. 2, can effectively use SV molecular markers to distinguish corn plant types with different nitrogen utilization efficiencies.

[0057] Example 4: Verifying the accuracy of the effect of SV variation on nitrogen use efficiency

[0058] To further verify the accuracy of the effect of SV variation on nitrogen use efficiency and the efficiency of SV molecular markers in distinguishing high and low nitrogen use efficiency materials, 516 genetically diverse maize inbred lines were genotyped. The genotyping results are detailed in Table 1. In addition, 302 of the materials were selected and their grain amino acid content was determined. The results are shown in the Appendix. Figure 5 As shown in Table 2, the test results show that compared with the low nitrogen utilization efficiency material (ASD1-TE), the high nitrogen utilization efficiency material (ASD1) shows a higher advantage in amino acid metabolism efficiency, and its lysine content is 10% higher than the former.

[0059] Table 1 Polymorphic sites in natural maize populations

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] Table 2 Results of amino acid content determination in 302 grains

[0067]

[0068]

[0069]

[0070] Example 5. Results of association analysis between the status of gene polymorphisms in natural populations and nitrogen use efficiency

[0071] In 2022, this study was conducted at the experimental site of Huazhong Agricultural University (located in Wuhan City, Hubei Province, including field and potted environments) and at the Xiangyang Experimental Base of Huazhong Agricultural University (located in Huangji Town, Xiangyang City, Hubei Province) for two consecutive years in 2023 and 2024. The experiment involved planting a series of diverse populations of maize inbred lines under high nitrogen and low nitrogen conditions, and measuring the nitrogen use efficiency of different maize varieties. The association analysis between plant height data and polymorphic loci was performed using GAPIT3 software. The mixed linear model combined with the population structure (MIM+(Q+K)) method was used for data analysis, and P<0.05 was used as the threshold for the significance level. For detailed results, see Figure 6 As shown in Table 3.

[0072] Table 3 Association analysis results between gene polymorphisms in natural populations and nitrogen use efficiency

[0073]

[0074] The association analysis results in Table 3 indicate that plant height differences between the two types of maize inbred lines within the diverse population of maize inbred lines reached significant levels (P < 0.05). Specifically, the nitrogen use efficiency of maize inbred lines I was higher than that of maize inbred lines II. Across the four environments, the nitrogen use efficiency of maize inbred lines I was 0.069, 0.021, 0.052, and 0.02 higher than that of maize inbred lines II, respectively. This study of diverse populations indicates that Type I is an excellent genotype for high nitrogen use efficiency in maize.

[0075] 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.

[0076] 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.

[0077] 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. An SV molecular marker associated with nitrogen use efficiency in maize, characterized by: The SV molecular marker is located on bases 6722060 to 6724624 of chromosome 5 of the maize B73 genome with a genome version number of GRAMENE-4.0, and its sequence is shown in SEQ ID NO.

3.

2. Use of the SV molecular marker according to claim 1 in breeding for nitrogen use efficiency of maize.

3. Use of the SV molecular marker according to claim 1 in identifying nitrogen utilization efficiency of corn.

4. Use of a reagent for specifically detecting the SV molecular marker of claim 1 in identifying and / or screening corn with different nitrogen use efficiencies, the reagent being a primer pair for specifically amplifying the SV molecular marker of claim 1, the primer pair comprising a forward primer ASD-F and a reverse primer ASD-R; The nucleotide sequence of the forward primer ASD-F is shown in SEQ ID NO.1; The nucleotide sequence of the reverse primer ASD-R is shown in SEQ ID NO.

2.

5. Use of the SV molecular marker or detection kit according to claim 1 in constructing a molecular identity card for maize, wherein the detection kit comprises a primer pair and a PCR amplification reagent, wherein the primer pair comprises a forward primer ASD-F and a reverse primer ASD-R; The nucleotide sequence of the forward primer ASD-F is shown in SEQ ID NO.1; The nucleotide sequence of the reverse primer ASD-R is shown in SEQ ID NO.

2.

6. A method for identifying the nitrogen use efficiency trait of corn, characterized in that: The steps include: Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using a primer pair to obtain a PCR amplification product; the PCR amplification product is subjected to gel electrophoresis and a judgment is made; the primer pair includes a forward primer ASD-F and a reverse primer ASD-R; The nucleotide sequence of the forward primer ASD-F is shown in SEQ ID NO.1; The nucleotide sequence of the reverse primer ASD-R is shown in SEQ ID NO.2; When the PCR amplification product shows a band at a length of 429 bp, the sample to be tested is a high nitrogen utilization type; when the PCR amplification product shows a band at a length of 3232 bp, the sample to be tested is a homozygous low nitrogen utilization efficiency type; When the PCR amplification product shows one band at the lengths of 429 bp and 3232 bp respectively, the sample to be tested is of heterozygous type.

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