SV molecular marker related to corn nitrogen absorption efficiency character and application of SV molecular marker

Abstract

The invention discloses an SV molecular marker related to a corn nitrogen absorption efficiency character and application thereof, the SV molecular marker is located in a 94937k-94941k base interval on a No. 1 chromosome of a corn B73 genome, and a corresponding primer and a detection kit are designed according to the SV molecular marker, so that prediction and screening of the corn nitrogen absorption efficiency character are realized, and the corn nitrogen absorption efficiency character is obtained. And a scientific basis is provided for cultivating corn inbred lines with different nitrogen absorption efficiency types.

Description

Technical Field

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

[0002] As one of the key "life elements" for plant growth, nitrogen has a very important impact on plant growth and development and yield formation. As corn is the main food crop in my country, the application of nitrogen fertilizer can effectively promote its leaf growth, increase chlorophyll content, and thus improve photosynthesis efficiency, ultimately increasing yield.

[0003] However, the utilization efficiency of nitrogen in the soil is generally low. According to a study by Raun and Johnson in 1999, for every 1000g of nitrogen input, only 330g can be absorbed by corn, and most of the remaining nitrogen will be lost through volatilization, leaching and other pathways, which not only causes a waste of resources, but also causes environmental pollution problems. Therefore, improving the absorption efficiency of soil nitrogen by corn is extremely critical to increasing corn yields, reducing the amount of nitrogen fertilizer applied, and promoting the development of green agriculture.

[0004] In terms of related gene research, existing results have shown that in an environment with sufficient nitrogen, knocking out the ZmNRT1.1B gene will cause a decrease in maize yield per plant; while under low nitrogen conditions, the maize yield per plant remains unchanged after knocking out the gene. In addition, overexpression of the ZmNRT1.1B gene can significantly increase the ear length and 100-grain weight of the excellent maize hybrid Xianyu 335 under low nitrogen conditions (Cao and Liu et al., 2023).

[0005] Genome structural variation (SV) refers to the change of long-length sequences and changes in positional relationships on the genome. It includes many types, such as insertion or deletion of long fragment sequences longer than 50bp (Big Indel), tandem repeats, chromosome inversions, sequence translocations within or between chromosomes, copy number variations (CNV), and more complex mosaic variations. Compared with other types of variation, SV has obvious differences in length, source, function, and impact. SV can detect structural variations of thousands of bases on the genome, and its markers show co-dominant genetic characteristics with good repeatability and stability.

[0006] Based on the above, how to use molecular marker technology to accurately predict the nitrogen absorption efficiency of corn 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 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 discloses a reagent for specifically detecting SV molecular markers related to the trait of nitrogen absorption efficiency of corn, and the reagent is a primer pair for specifically amplifying SV molecular markers related to the trait of nitrogen absorption efficiency 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 comprising the primer pair according to claim 1 or 2 and a PCR amplification reagent.

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

[0015] Application of reagents for specific detection of SV molecular markers related to maize nitrogen absorption efficiency traits 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 trait of nitrogen absorption efficiency of corn or a detection kit containing a primer pair and a PCR amplification reagent for specifically amplifying SV molecular markers related to the trait of nitrogen absorption efficiency of corn in constructing a molecular ID 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 primer pair described in claim 1 or 2 to obtain a PCR amplification product; performing gel electrophoresis on the PCR amplification product and making a judgment;

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

[0021] The beneficial effects of the above technical solution are: the research team found an SV molecular marker with nitrogen absorption efficiency response in the 94937k-94941k base interval on chromosome 1 of the corn B73 genome, and the SV molecular marker is significantly correlated with the nitrogen absorption efficiency trait of corn; the present invention designs corresponding primers and detection kits based on SV molecular markers, thereby realizing the prediction and screening of corn nitrogen absorption efficiency traits, and providing a scientific basis for breeding corn inbred lines with different nitrogen absorption efficiency types. At the same time, there is no need to consider the growth period and tissue type of corn during the detection process, 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 ideal corn plant types and improve breeding efficiency.

[0022] Based on the above technical advantages, the present invention also provides a method for identifying the nitrogen absorption efficiency trait of corn, comprising the following steps: using the genomic DNA of the 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; performing gel electrophoresis on the PCR amplification product and making a judgment. Experiments have shown that the SV marker provided by the present invention and the primers designed based on the marker can accurately detect the nitrogen absorption efficiency trait of corn and effectively distinguish the types of genotypes. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[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 that leaf biomass and plant biomass of the AMP population under low nitrogen conditions were significantly different between hap0 and hap1. 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 by purchasing from the market. The experimental methods used in the following examples are 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, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or combinations thereof.

[0029] It should also be understood that the term “and / or” used in the specification and appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0030] References to "one embodiment" or "some embodiments" etc. described in the specification of this application mean that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, the statements "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in different places in this specification do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[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 solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 94937k-94941k base interval 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 name of the genome database is: 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 found that the key gene ZmNRT1.1B controlling nitrogen absorption of maize has two types of promoters in the natural maize population, one is the promoter with transposon insertion and the other is the promoter without transposon insertion. Based on this discovery, the research team carried out the following research:

[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 absorption 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 genotype fragments with different nitrogen absorption efficiency with SV molecular marker insertion, and perform haplotype typing on whether the gene promoter in 504 materials of AMP population contains transposon. The process is as follows:

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

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

[0040] The PCR amplification system was calculated as 20 μL and included: 2× TaqMasterMix 10ul, 60-300ng / μL genomic DNA template 1μL, 10μM 1F 1μL, 10μM 1R 1μL, and ddH2O to 20μL;

[0041] The PCR amplification program was as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 60 sec / kb, 34 cycles; 72°C extension for 5 min, 12°C for 2 min. 2×Taq MasterMix was purchased from Nanjing Novozyme 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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[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, through the LUC experiment, the ZmNRT1.1B promoter with transposon (haplotype hap0) was connected to the pGreen II 0800 vector, and the ZmNRT1.1B promoter without transposon (haplotype hap1) was selected and also connected to the pGreen II 0800 vector, and the protoplasts of yellowing corn seedlings were transformed, and the enzyme activity was determined. The results showed that the LUC activity of the promoter with transposon insertion was lower than that of the promoter without transposon insertion, and the gene expression level of the promoter with transposon insertion was lower than that of the promoter without transposon insertion.

[0052] In summary, it can be seen that hap0 with a fragment size of 3790 bp is a genotype with low nitrogen absorption efficiency, and hap1 with a fragment size of 481 bp is a genotype with high nitrogen absorption efficiency. At the same time, it can be seen that the primer set shown in SEQ ID NO.1 to SEQ ID NO.2 provided by the present invention can effectively use SV molecular markers to distinguish corn plant types with different corn nitrogen utilization 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. 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] It can be seen from Tables 2 and 3 that the root expression levels and leaf expression levels of hap0 material are lower than those of hap1 material, and the differences in root expression levels and leaf expression levels of the two types formed by AMP populations are significant (P<0.05). The difference in expression levels shows that the nitrogen absorption efficiency of hap0 material is lower than that of hap1 material.

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

[0069] The AMP population was planted in the field in Wuhan, and 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 AMP population under low nitrogen conditions

[0071]

[0072]

[0073]

[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] As shown in Tables 4 and 5, under low nitrogen conditions, the leaf biomass and plant biomass of hap0 material were lower than those of hap1 material, and the differences in leaf biomass and plant biomass of the two types formed by the AMP population reached a significant level (P<0.05), and the nitrogen absorption efficiency of hap0 material was lower than that of hap1 material. The study of the diversity population showed that type hap1 is an excellent genotype of high nitrogen absorption efficiency of corn.

[0077] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that numerous changes, modifications, substitutions and variations may be made to the examples without departing from the principles and spirit of the invention, the scope of the invention being defined by the appended claims and their equivalents.

[0078] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant descriptions 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 the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included in the protection scope of the present invention.

Claims

1. A reagent for specifically detecting SV molecular markers related to maize nitrogen absorption efficiency traits, characterized in that: The reagent is a primer pair used for specifically amplifying SV molecular markers related to the nitrogen absorption efficiency trait of corn.

2. The reagent according to claim 1, characterized in that: 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.

3. A detection kit, characterized in that: Contains the primer pair according to claim 1 or 2 and a PCR amplification reagent.

4. Use of the reagent described in claims 1-2 in breeding for nitrogen absorption efficiency of corn.

5. Use of the reagent according to claims 1-2 in identifying the nitrogen absorption efficiency of corn.

6. Use of the reagent according to any one of claims 1 to 2 in screening corns with different nitrogen absorption efficiencies.

7. Use of the reagents described in claims 1-2 or the detection kit described in claim 3 in constructing a molecular identity card of corn.

8. 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 the primer pair described in claim 1 or 2 to obtain a PCR amplification product; 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 a 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 a high nitrogen absorption type.

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