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

Abstract

The present invention discloses an SV molecular marker related to the nitrogen absorption efficiency trait of corn and its application. The SV molecular marker is located in the 94937k to 94941k base interval on chromosome 1 of the corn B73 genome. Corresponding primers and detection kits are designed based on the SV molecular marker, thereby realizing the prediction and screening of the nitrogen absorption efficiency trait of corn, and providing a scientific basis for breeding corn inbred lines with different nitrogen absorption 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 absorption efficiency trait of corn and an application thereof. Background Art

[0002] Nitrogen, as one of the key "vital elements" for plant growth, has a crucial impact on plant development and yield formation. For corn, a staple food crop in my country, nitrogen fertilizer can effectively promote leaf growth, increase chlorophyll content, and thus improve photosynthesis efficiency, ultimately increasing yield.

[0003] However, soil nitrogen utilization efficiency is generally low. According to a 1999 study by Raun and Johnson, for every 1,000g of nitrogen applied, only 330g is absorbed by corn, while the remainder is lost through volatilization and leaching. This not only wastes resources but also causes environmental pollution. Therefore, improving corn's efficiency in soil nitrogen absorption is crucial for increasing corn yields, reducing nitrogen fertilizer application, and promoting green agriculture.

[0004] Research on related genes has shown that knocking out the ZmNRT1.1B gene in nitrogen-sufficient environments reduces maize yield per plant; however, under low nitrogen conditions, knocking out the gene maintains yield per plant. Furthermore, overexpressing the ZmNRT1.1B gene significantly increases ear length and 100-kernel weight in the elite maize hybrid Xianyu 335 under low nitrogen conditions (Cao and Liu et al., 2023).

[0005] Genomic structural variation (SV) refers to changes in the length and position of large sequences in the genome. It includes various types, such as long sequence insertions or deletions (Big Indels) longer than 50 bp, 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 codominant inheritance characteristics, with good repeatability and stability.

[0006] In view of the above, how to accurately predict the nitrogen absorption efficiency of corn with the help of molecular marker technology and cultivate corn varieties with high nitrogen absorption efficiency has become a technical problem that urgently needs to be overcome in this field. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an SV molecular marker related to the nitrogen absorption efficiency trait of corn and an application thereof.

[0008] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0009] The invention relates to a reagent for specifically detecting SV molecular markers related to the nitrogen absorption efficiency trait of corn. The reagent is a primer pair for specifically amplifying SV molecular markers related to the nitrogen absorption efficiency trait of corn.

[0010] Further optimized, the primer pair includes a forward primer 1F and a reverse primer 1R;

[0011] The nucleotide sequence of the forward primer 1F is shown in SEQ ID NO.1;

[0012] The nucleotide sequence of the reverse primer 1R is shown in SEQ ID NO.2.

[0013] A detection kit contains the above primer pair and PCR amplification reagent.

[0014] Application of reagents for specifically detecting SV molecular markers related to maize nitrogen uptake efficiency traits in maize nitrogen uptake efficiency breeding.

[0015] Application of reagents for specific detection of SV molecular markers related to maize nitrogen absorption efficiency in the identification of maize nitrogen absorption efficiency.

[0016] Application of reagents for specifically detecting SV molecular markers related to the nitrogen absorption efficiency trait of corn in screening corns with different nitrogen absorption efficiencies.

[0017] The invention relates to the use of a reagent for specifically detecting SV molecular markers related to the nitrogen absorption efficiency trait of corn or a detection kit containing a primer pair and a PCR amplification reagent for specifically amplifying SV molecular markers related to the nitrogen absorption efficiency trait of corn in constructing a molecular identity card of corn.

[0018] A method for identifying the nitrogen absorption efficiency trait of corn comprises the following steps:

[0019] Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using the above primer pair to obtain a PCR amplification product; the PCR amplification product is subjected to gel electrophoresis and a judgment is made;

[0020] When the PCR amplification product shows a band at a length of 3790 bp, the sample to be tested is of the low nitrogen absorption type; when the PCR amplification product shows a band at a length of 481 bp, the sample to be tested is of the high nitrogen absorption type.

[0021] The beneficial effects of adopting the above technical solution are as follows: the research team discovered an SV molecular marker with a nitrogen absorption efficiency response in the 94937k-94941k base interval on chromosome 1 of the maize B73 genome. This SV molecular marker is significantly correlated with the nitrogen absorption efficiency trait of maize. The present invention designed corresponding primers and detection kits based on the SV molecular marker, thereby realizing the prediction and screening of the maize nitrogen absorption efficiency trait, providing a scientific basis for breeding maize inbred lines with different nitrogen absorption efficiency types. At the same time, the detection process does not need to consider the growth period and tissue type of maize, nor does it require field phenotyping. The genotype information of the sample can be accurately and quickly obtained, which is beneficial to accelerate the germplasm innovation of ideal maize plant types and improve breeding efficiency.

[0022] Based on the above technical advantages, the present invention also provides a method for identifying the nitrogen uptake 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 uptake efficiency trait in maize and effectively distinguish genotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the molecular detection results of the primer set in materials with differential expression of the ZmNRT1.1B gene;

[0024] Figure 2 Schematic diagram of LUC experiment verifying that ZmNRT1.1B promoter TE insertion reduces the expression of the gene, where A is the schematic diagram of gene construction and B is the bar graph of gene expression levels;

[0025] Figure 3 This is a schematic diagram showing that the expression levels of AMP population materials are significantly different between hap0 and hap1;

[0026] Figure 4 Schematic diagram showing significant differences in leaf biomass and plant biomass between hap0 and hap1 in the AMP population under low nitrogen conditions. DETAILED DESCRIPTION

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] This research team discovered an SV molecular marker that responds to nitrogen absorption efficiency in the base interval of 94937k to 94941k on chromosome 1 of the maize B73 genome. The version of the maize B73 genome is Zm-B73-REFERENCE-GRAMENE-4.0; the download address of the maize B73 genome is https: / / download.maizegdb.org / Zm-B73-REFERENCE-GRAMENE-4.0 / , and the genome database is named: MAIZEGDB; the maize B73 genome is publicly available in DOI:10.1038 / nature22971.

[0034] Example 1: PCR amplification, electrophoresis detection, and genotyping

[0035] The present invention team discovered in natural maize populations that the key gene ZmNRT1.1B, which controls maize nitrogen uptake, has two types of promoters: one with transposon insertions and the other without. Based on this discovery, the research team conducted the following studies:

[0036] Based on the presence or absence of transposon insertion in the promoter of gene ZmNRT1.1B, a specific primer set for identifying the nitrogen uptake efficiency trait of maize was designed. The specific primer set includes a forward primer 1F (SEQ ID NO.1) and a reverse primer 1R (SEQ ID NO.2);

[0037] Forward primer 1F and reverse primer 1R can specifically amplify fragments of different nitrogen absorption efficiency genotypes with SV molecular marker insertions. Haplotype typing was performed to determine whether the gene promoters in 504 materials of the AMP population contained transposons. The process is as follows:

[0038] A. The CTAB method was used to extract maize genomic DNA from different extreme materials;

[0039] B. PCR amplification: Using the maize genomic DNA extracted in step A as a template, PCR amplification was performed using primer pair 1F and 1R (SEQ ID NO. 1 to SEQ ID NO. 2) in step (1) to obtain a PCR amplification product;

[0040] The PCR amplification system (20 μL) included: 10 μL of 2× TaqMasterMix, 1 μL of 60–300 ng / μL genomic DNA template, 1 μL of 10 μM 1F, 1 μL of 10 μM 1R, and ddH2O to 20 μL.

[0041] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; 34 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 60 sec / kb; extension at 72°C for 5 min and 12°C for 2 min. 2× Taq MasterMix was purchased from Nanjing Novozymes Biotech Co., Ltd.

[0042] PCR agarose gel electrophoresis detection: Use 1% agarose gel to detect the PCR amplification product in step B.

[0043] Depend on Figure 1 It can be seen that after electrophoresis detection of the PCR amplification product, there is a band at 3790 bp or 481 bp. The fragment size of 3790 bp corresponds to the genotype hap0; the fragment size of 481 bp corresponds to the genotype hap1.

[0044] The typing results are shown in Table 1.

[0045] Table 1 Haplotype typing results of AMP population materials

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[0047]

[0048]

[0049]

[0050] From the results in Table 1, we can see that 30 materials in the 504 AMP populations have transposon insertions, belonging to hap0, and 474 materials have no transposon insertions, belonging to hap1.

[0051] like Figure 2 As shown in the figure, through the LUC experiment, the ZmNRT1.1B promoter with a transposon (haplotype hap0) was connected to the pGreen II 0800 vector, and the ZmNRT1.1B promoter without a transposon (haplotype hap1) was also connected to the pGreen II 0800 vector. The protoplasts of yellowing maize seedlings were transformed and the enzyme activity was determined. The results showed that the LUC activity of the promoter with a transposon insertion was lower than that of the promoter without a transposon insertion, and the gene expression level of the promoter with a transposon insertion was lower than that of the promoter without a transposon insertion.

[0052] In summary, the hap0 fragment (3790 bp) represents a genotype with low nitrogen uptake efficiency, while the hap1 fragment (481 bp) represents a genotype with high nitrogen uptake efficiency. Furthermore, the primer sets provided by the present invention, as shown in SEQ ID NOs. 1 to 2, can effectively utilize SV molecular markers to distinguish maize plant types with different nitrogen use efficiencies.

[0053] Example 2: Analysis of expression levels of materials of two haplotypes, hap0 and hap1

[0054] The expression levels of 461 samples of hap0 and hap1 haplotypes in the AMP population were analyzed, and the results are shown in Tables 2, 3 and Figure 3 shown.

[0055] Table 2 Expression results of AMP population materials between hap0 and hap1

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[0064] Note: NA indicates missing data

[0065] Table 3 Correlation analysis results of expression levels of AMP population materials between hap0 and hap1

[0066]

[0067] As shown in Tables 2 and 3, the root and leaf expression levels of the hap0 material were lower than those of the hap1 material. The differences in root and leaf expression levels of the two types of AMP populations reached a significant level (P<0.05). The differences in expression levels showed that the nitrogen absorption efficiency of the hap0 material was lower than that of the hap1 material.

[0068] Example 3: Analysis of leaf biomass and plant biomass of AMP population materials at maturity

[0069] By planting AMP populations in Wuhan fields, the leaf biomass and plant biomass of 190 population materials at maturity were investigated. The results are shown in Tables 4, 5 and Figure 4 .

[0070] Table 4 Statistical results of leaf biomass and plant biomass in different haplotypes of hap0 and hap1 in the AMP population under low nitrogen conditions

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[0072]

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[0074] Table 5 Results of association analysis of leaf biomass and plant biomass in different haplotypes of hap0 and hap1 in the AMP population under low nitrogen conditions

[0075]

[0076] Tables 4 and 5 show that under low nitrogen conditions, the leaf and plant biomass of the hap0 accession were lower than those of the hap1 accession. The differences in leaf and plant biomass between the two types of AMP populations reached significant levels (P < 0.05). The nitrogen absorption efficiency of the hap0 accession was lower than that of the hap1 accession. Studies of diverse populations indicate that the hap1 accession is an excellent genotype for high nitrogen absorption efficiency in maize.

[0077] 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.

[0078] 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.

[0079] 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. Application of a primer pair in breeding for nitrogen absorption efficiency in maize, the primer pair comprising a forward primer 1F and a reverse primer 1R; The nucleotide sequence of the forward primer 1F is shown in SEQ ID NO.1; The nucleotide sequence of the reverse primer 1R is shown in SEQ ID NO.

2.

2. Application of a primer pair in identifying nitrogen absorption efficiency of corn, the primer pair comprising a forward primer 1F and a reverse primer 1R; The nucleotide sequence of the forward primer 1F is shown in SEQ ID NO.1; The nucleotide sequence of the reverse primer 1R is shown in SEQ ID NO.

2.

3. Application of a primer pair in screening corn with different nitrogen absorption efficiencies, the primer pair comprising a forward primer 1F and a reverse primer 1R; The nucleotide sequence of the forward primer 1F is shown in SEQ ID NO.1; The nucleotide sequence of the reverse primer 1R is shown in SEQ ID NO.

2.

4. Application of a detection kit in constructing a molecular identity card for maize, the detection kit comprising a primer pair and a PCR amplification reagent, the primer pair comprising a forward primer 1F and a reverse primer 1R; The nucleotide sequence of the forward primer 1F is shown in SEQ ID NO.1; The nucleotide sequence of the reverse primer 1R is shown in SEQ ID NO.

2.

5. A method for identifying the nitrogen absorption 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 primer pair includes a forward primer 1F and a reverse primer 1R; The nucleotide sequence of the forward primer 1F is shown in SEQ ID NO.1; The nucleotide sequence of the reverse primer 1R is shown in SEQ ID NO.2; Performing gel electrophoresis on the PCR amplification product and making a judgment; When the PCR amplification product shows a band at a length of 3790 bp, the sample to be tested is of the low nitrogen absorption type; when the PCR amplification product shows a band at a length of 481 bp, the sample to be tested is of the high nitrogen absorption type.

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