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

Abstract

The invention discloses an SV molecular marker related to a corn nitrogen utilization efficiency character and application of the SV molecular marker. The SV molecular marker is located on the 6722060-6724624 basic group of the fifth chromosome of a corn B73 genome with the genome version number of GRAMENE-4. 0, and the sequence of the SV molecular marker is shown as SEQ ID NO.3. The invention further discloses a method for preparing the SV molecular marker related to the corn nitrogen utilization efficiency character and application of the SV molecular marker related to the corn nitrogen utilization efficiency character. The corresponding primers and the detection kit are designed according to the SV molecular marker, so that prediction and screening of the corn nitrogen utilization efficiency character are realized, and a scientific basis is provided for cultivation of corn inbred lines with different nitrogen utilization 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 utilization efficiency trait of corn and application thereof. Background Art

[0002] Corn (Zea mays L.) is the largest food crop in my country. Its stable and high yield is one of the key challenges facing the scientific breeding community. Nitrogen use efficiency occupies an extremely important position among the many agronomic traits of corn. In the corn production process, corn with low nitrogen use efficiency is difficult to absorb excessive nitrogen fertilizer, which not only causes a waste of 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 corn is closely linked to the realization of green and efficient production. Improving the nitrogen use efficiency of corn is of great significance. It can not only reduce the application of nitrogen fertilizer 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 corn. For example, Zhang et al. successfully located 73 genetic loci related to nitrogen metabolism enzyme activity with the help of offspring separation populations of B73 and Mo17, 10 of which belong to key enzyme loci 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, the existing technology mostly uses field phenotyping when marking corn nitrogen utilization traits. This method has obvious disadvantages and requires a lot of manpower and material resources.

[0004] Genome structural variation (Structure variation, SV) refers to the changes in the sequence length and position relationship of the genome. It covers many types, including long fragment sequence insertion or deletion (Big Indel) with a length of more than 50bp, tandem repeats, chromosome inversion, sequence translocation within or between chromosomes, copy number variation (Copy Number Variations, CNV) and more complex mosaic variation. Compared with other types of variation, SV has significant differences in length, source, function and impact. SV can detect structural variations of thousands of bases in the genome, and its markers show 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 application thereof.

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

[0008] A SV molecular marker related to the nitrogen utilization efficiency trait of corn, the SV molecular marker is located on the 6722060-6724624 bases of chromosome 5 of the corn B73 genome with the genome version number GRAMENE-4.0, and its sequence is shown in SEQID NO.3.

[0009] A reagent for specifically detecting SV molecular markers located on bases 6722060 to 6724624 of chromosome 5 of the corn B73 genome with a genome version number of 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 a genome version number of 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 markers located at bases 6722060 to 6724624 on chromosome 5 of the maize B73 genome with genome version number GRAMENE-4.0 in breeding for maize nitrogen use efficiency.

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

[0017] Use of the reagent for specifically detecting the SV molecular marker according to claim 1 as described in 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 6722060-6724624 of chromosome 5 of the corn B73 genome with a genome version number of GRAMENE-4.0 or a detection kit containing the primer pair and PCR amplification reagent described in claim 3 or 4 in constructing a corn molecular ID card.

[0019] A method for identifying the nitrogen utilization 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 429bp, the sample to be tested is of the high nitrogen utilization type; when the PCR amplification product shows a band at a length of 3232bp, the sample to be tested is of the homozygous low nitrogen utilization efficiency type; when the PCR amplification product shows a band at the lengths of 429bp and 3232bp respectively, the sample to be tested is of the 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 markers, thereby realizing the prediction and screening of the nitrogen utilization efficiency trait of corn, and providing a scientific basis for breeding corn inbred lines of different nitrogen utilization efficiency types. At the same time, there is no need to consider the growth period and tissue type of corn during the detection process, and there is no need to conduct field phenotypic investigations, and 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 of corn and improve breeding efficiency.

[0023] Based on the above technical advantages, the present invention also provides a method for identifying the nitrogen utilization 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 utilization efficiency trait of corn and effectively distinguish the pure heterozygous type of the genotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the results of the genome-wide association analysis of nitrogen utilization 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 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 421 maize diversity populations under high nitrogen and low nitrogen conditions; C is a Manhattan plot of the genome-wide association analysis of 421 maize diversity populations based on the expression of related genes;

[0025] Figure 2 This is a 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 It is a bar graph of the investigation results of nitrogen utilization efficiency of different maize inbred line materials based on molecular detection typing in Example 4;

[0029] Figure 6 This is a schematic diagram of the results of the association analysis between the status of the gene polymorphism sites in the natural population and the 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 by purchasing from the market. The experimental methods used in the following examples are 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, 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.

[0032] 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.

[0033] 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.

[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 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.

[0036] The research team of the present invention provides an SV molecular marker related to the nitrogen use 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; the download address of the corn B73 genome is https: / / download.maizegdb.org / Zm-B73-REFERENCE-GRAMENE-4.0 / , and the name of the genome database is: MAIZEGDB; the corn B73 genome is disclosed 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 diversity populations were planted in the experimental field in Xiangyang, Hubei, and nitrogen use efficiency was measured in July and September, and three nitrogen use efficiency phenotypic data were collected. Among them, the 421 diversity populations and the selected extreme nitrogen use efficiency phenotype maize materials were selected from the National Key Laboratory of Crop Genetic Improvement of Huazhong Agricultural University or the publicly available genome resequencing maize cultivars on the Internet (for details, please refer to http: / / maizego.org / Resources.html and https: / / download.maizegdb.org).

[0040] Based on the 421 maize inbred lines whose genomes have been resequenced by the State Key Laboratory of Crop Genetic Improvement of 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 attached. Figure 1 It can be seen that the nitrogen utilization efficiency data of the diverse maize populations 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 6717165-6728461 base sequence interval of chromosome 5. The results are shown in the attached Figure 2 , where the black marks are large insertions and deletions. We selected Zheng 58 and Chang 7-2, parents of the widely planted commercial hybrid Zhengdan 968, Jing 92 and Jing 724, parents of Jingke 968, a temperate material Ye 478, and a tropical material CML103. Figure 2The sequencing comparison results show that the materials with high nitrogen utilization efficiency (Jing 92, Chang 7-2 and CML103) have a fragment of about 2.5kb inserted in them compared with the materials with low nitrogen utilization efficiency (Jing 724, Zheng 58 and Ye 478). The fragment of about 2.5kb is recorded as SV molecular marker; the variation type is a large fragment insertion and deletion variation in SV molecular marker. That is, the SV molecular marker is directly related to the nitrogen utilization efficiency trait of corn. The material with the insertion is likely to show the low nitrogen utilization efficiency trait type, while the material without the 2.5kb insertion is likely to show the high nitrogen utilization efficiency trait type.

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

[0044] In order to further determine the accuracy of the effect of SV molecular markers on maize nitrogen use efficiency traits in Example 1, the physical location information of SV molecular markers was determined based on the maize B73 genome GRAMENE-4.0 version as: the nucleotide sequence shown in bases 6722060 to 6724624 of maize chromosome 5. The sequence is located in the intron of the ASD1 gene, and the ASD1 gene is an amino acid metabolism gene among multiple species. The extreme materials CIMBL144, CY72, CML323, CIMBL16, TY8, 5213, GEMS27 and GEMS2 with low nitrogen utilization efficiency and the extreme materials D047, GEMS12, CIMBL95, H21, CIMBL152, CIMBL51 and GEMS35 with high nitrogen utilization efficiency were selected, and the above extreme materials were planted. When they were planted to 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 different extreme materials was extracted by the kit method (RNA extraction kit was purchased from Nanjing Novozyme Biotechnology Co., Ltd.), and the expression level of ASD1 was detected by qRT-PCR. The results are shown in the attached Figure 3 .

[0045] By the attached Figure 3 It can be seen that the expression 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. It can be seen that the insertion and deletion variation of about 2.5 kb in Example 1 regulates the nitrogen use efficiency trait of corn by regulating its expression. Therefore, the large insertion and deletion variation of about 2.5 kb is a key SV molecular marker affecting the nitrogen use efficiency trait of corn.

[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, and the specific primer set included 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 insertion. 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 are used as the test materials, and the steps are as follows:

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

[0052] B. PCR amplification: using the corn 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) to perform PCR amplification to obtain a PCR amplification product;

[0053] The PCR amplification system was based on 20 μL, specifically: 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 ddHO. 2 O;

[0054] The PCR amplification program was as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 210 s, 34 cycles; 72°C extension for 5 min, 10°C storage for 2 min. 2×Taq Plus Master Mix was purchased from Nanjing Novozyme 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 4In the figure, 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 It can be seen that after electrophoresis detection, the PCR amplification product of low nitrogen utilization has only one band at 3232 bp, while the PCR amplification product of high nitrogen utilization efficiency extreme material has one band at 429 bp. 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.

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

[0058] In order 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 shown in Table 1. In addition, 302 materials were selected and their grain amino acid content was measured. The results are shown in 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 loci 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 polymorphism sites in natural populations and nitrogen utilization 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 corn inbred lines under high-nitrogen and low-nitrogen conditions, and the nitrogen utilization efficiency of different corn varieties was measured. The GAPIT3 software was used to perform association analysis on plant height data and polymorphic loci. 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 Results of association analysis between gene polymorphisms in natural populations and nitrogen use efficiency

[0073]

[0074] The results of association analysis in Table 3 show that the differences in plant height of the two types formed by the diversity population composed of maize inbred lines reached a significant level (P<0.05). Among them, the nitrogen utilization efficiency of maize type I was higher than that of maize type II. In the four environments, the nitrogen utilization efficiency of maize inbred line materials of type I was 0.069, 0.021, 0.052, and 0.02 higher than that of maize II, respectively. The study of the diversity population showed that type I is an excellent genotype for high nitrogen utilization efficiency of maize.

[0075] 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.

[0076] 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.

[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 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. An SV molecular marker associated with the nitrogen use efficiency trait of maize, characterized in that: 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. A reagent for specifically detecting the SV molecular marker according to claim 1.

3. The reagent according to claim 2, characterized in that: The reagent for specifically detecting the SV molecular marker according to claim 1 is a primer pair for specifically amplifying the SV molecular marker according to claim 1.

4. The reagent according to claim 3, characterized in that: 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.

5. A detection kit, characterized in that: Contains the primer pair according to claim 3 or 4 and a PCR amplification reagent.

6. Use of the SV molecular marker described in claim 1 in breeding for nitrogen utilization efficiency of corn.

7. Use of the SV molecular marker described in claim 1 in the identification of nitrogen utilization efficiency of corn.

8. 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 efficiency.

9. Use of the SV molecular marker described in claim 1 or the detection kit described in claim 5 in constructing a molecular identity card of maize.

10. A method for identifying the nitrogen utilization 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 3 or 4 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 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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