Maize dwarfing gene br2-d2308 as well as molecular marker and application thereof

Abstract

The invention discloses a corn dwarfing gene br2-d2308 as well as a molecular marker and application thereof, and relates to the technical field of corn breeding, the br2-d2308 gene is obtained after the 1276th basic group of the fifth exon of a corn Br2 gene is mutated into A from G, and the nucleotide sequence of the fifth exon of the br2-d2308 gene is as shown in SEQ ID NO.1. The molecular marker of the corn dwarfing gene br2-d2308 and the application of the molecular marker of the corn dwarfing gene br2-d2308. Researches find that the plant height and the ear height of the mutant d2308 are remarkably reduced compared with those of a wild type B73, the yield loss of the mutant d2308 is smaller than that of other corn dwarfing mutants, other obvious bad characters do not exist, a new lodging-resistant dwarfing density-tolerant high-yield corn variety can be created by utilizing the br2-d2308 gene, an existing variety can be improved, and the corn dwarfing mutant d2308 has a wide application prospect. The method has important breeding application value. The invention also develops a dCAPS marker based on the corn br2-d2308 gene, and the dCAPS marker can effectively distinguish dwarfing improved and non-dwarfing corn plants, and can improve the genetic improvement efficiency of dwarfing density-tolerant corn varieties.

Description

Technical Field

[0001] The invention relates to the technical field of corn breeding, and in particular to a corn dwarfing gene br2-d2308 and a molecular marker and application thereof. Background Art

[0002] Corn is an important crop for food, feed and industrial raw materials in my country. With the rapid development of animal husbandry and processing industries, its demand is showing a growing trend. The improvement of corn yield is closely related to planting density. Compared with developed countries abroad, my country's corn planting density is generally low, which seriously restricts the improvement of corn yield. Plant height is one of the key plant traits that determine corn planting density. Dwarfing breeding can enable plants to maintain good photosynthesis efficiency and lodging resistance under high-density planting conditions. Identifying excellent dwarfing genes and carrying out the selection and promotion of new dwarfing varieties are important ways to improve corn planting density and yield.

[0003] Plant height is a complex quantitative trait controlled by multiple genes. Currently, more than 60 maize dwarfing genes have been reported at home and abroad. These genes are mainly involved in the synthesis, metabolism and signal transduction of different types of hormones, including gibberellin (GA), auxin (IAA), brassinosteroids (BR), etc. Among the reported dwarfing genes, there are still few that can be directly applied to maize dwarfing breeding. The main reason is that many of these genes show excessive reduction in plant height or even extreme dwarfing, large yield losses, and more linkage drags of undesirable traits. The use of biological breeding technologies such as gene mutagenesis and gene editing to discover excellent dwarfing genes and excellent allelic variations of known functional genes is an important research content of current dwarfing breeding.

[0004] Auxin is a very important class of plant hormones, and its synthesis, metabolism, signal transduction, and polar transport are encoded by a multigene family. Brachytic2 (Br2) is the first dwarf gene discovered in maize. This gene plays an important role in the auxin transport process. Its functional loss leads to abnormal polar transport of auxin in the internode meristem, which inhibits cell elongation. The main characteristics of Br2 gene-related mutants are a significant reduction in plant height, especially the shortening of internodes below the ear. Due to its small impact on yield, it has become one of the dwarf genes with great potential for breeding applications. In recent years, multiple allelic variations of Br2 have been discovered using various technologies and methods, and these mutants also show different phenotypes. In previous studies, we also screened an allelic mutation d129 (patent number CN202210885719.2) of the Br2 gene through EMS mutagenesis. Its plant height and ear height were significantly reduced, and the field plant type was excellent, but it had a significant impact on grain weight and yield. Therefore, further creation and selection of excellent allelic variations of Br2 will provide excellent genetic resources for corn dwarf breeding, and significantly promote the improvement of corn planting density and yield level. Summary of the invention

[0005] The purpose of the present invention is to provide a maize dwarfing gene br2-d2308 and its molecular marker and application. The present invention achieves the above purpose through the following technical solutions:

[0006] The invention provides a corn dwarfing gene br2-d2308. The br2-d2308 gene is obtained by mutating the 1276th base of the 5th exon of the corn Br2 gene from G to A, and the mutation causes the encoded amino acid to mutate from glycine (G) to arginine (R). The nucleotide sequence of the 5th exon of the br2-d2308 gene is shown in SEQ ID NO.1.

[0007] The present invention also provides an application of a corn dwarfing gene br2-d2308 in corn molecular breeding.

[0008] A further improvement is to hybridize and backcross the corn germplasm to be improved with the corn germplasm containing the corn dwarfing gene br2-d2308, screen the offspring containing the relevant traits of the corn germplasm to be improved and the corn dwarfing gene br2-d2308, and obtain new dwarfed improved corn germplasm.

[0009] A further improvement is that, using gene editing technology, the 1276th base of the 5th exon of the Br2 gene in the genome of the corn to be improved is mutated from G to A, so as to obtain a dwarf strain of the corn germplasm to be improved.

[0010] The present invention also provides a molecular marker based on the maize dwarf gene br2-d2308, wherein the molecular marker is a dCAPS marker, and the dCAPS marker comprises an amplification primer for amplifying a nucleotide sequence containing the 1276th base of the 5th exon of the maize Br2 gene, wherein the base type of the 1276th base is G / A;

[0011] Also included are restriction enzymes for base typing verification.

[0012] A further improvement is that the restriction endonuclease is ApaI.

[0013] A further improvement is that the nucleotide sequence of the amplification primer is:

[0014] SEQ ID NO.7: M2308-F: 5'-TGCTGCAGAAGATGTTCATGAAGG-3';

[0015] SEQ ID NO. 8: M2308-R: 5'-CCGTAGCCCGCTGCCGGCGATGGGCC-3'.

[0016] The present invention also provides an application of the above-mentioned molecular marker in assisting the breeding of dwarf corn varieties.

[0017] The further improvement is that the genomic DNA of the corn germplasm to be tested is used as a template, the target sequence is amplified by PCR using the amplification primers labeled with dCAPS, the PCR amplification product is digested by the restriction endonuclease ApaI, and the genotyping of the digestion product is performed to obtain the molecular marker type of the corn germplasm to be tested;

[0018] The plant height of the tested corn germplasm or variety with the molecular marker type of A is significantly shorter than the plant height of the tested corn with the molecular marker type of G.

[0019] The present invention has the following beneficial effects: by analyzing the plant types of mutant d2308 and wild-type B73 plants, it can be found that the mutant d2308 has a significantly reduced plant height relative to the wild-type B73, and the plant type is compact. In addition, compared with some other corn dwarf mutants, the mutant d2308 of the present invention has very small yield loss and fewer adverse traits. The br2-d2308 gene can be used to create a new high-yield corn variety that is resistant to lodging, dwarfing and densely resistant, and to improve existing varieties, which has important breeding application value. In addition, the present invention also develops a dCAPS marker based on the corn dwarf gene br2-d2308. The amplification product obtained by PCR amplification of the dCAPS-marked amplification primer is subjected to enzyme digestion and then electrophoresis detection bands are detected. The band type difference between the control plant and the dwarf mutant is obvious, indicating that the dCAPS marker can effectively distinguish between dwarfing improved and non-dwarfing corn plants, and can improve the efficiency of genetic improvement of dwarfing and densely resistant corn germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Phenotypic analysis of the dwarf mutant d2308 ( Figure 1 A is the phenotypic analysis of B73 maize inbred line and dwarf mutant d2308; Figure 1 BC is the comparison of plant height and ear height of B73 and d2308 respectively; Figure 1 D is the comparison of stem diameters of the third internode above ground between B73 and d2308; Figure 1 EG are the comparison of leaf length, leaf width and leaf angle between B73 and d2308 respectively; S: ear leaf; S+1: the first leaf above the ear; S-1: the first leaf below the ear; S+5: the second leaf from the bottom);

[0021] Figure 2 Analysis of yield-related traits of dwarf mutant d2308 ( Figure 2 A is the comparison of ear phenotypes between the B73 maize inbred line and the dwarf mutant d2308; Figure 2 B is the phenotypic comparison of grain length (I), grain width (II) and grain thickness (III) between B73 and d2308; Figure 2 CJ is the statistics of ear length, number of kernels per row, kernel weight per ear, seed yield, 100-kernel weight, kernel thickness, kernel length and kernel width of B73 and d2308, respectively;

[0022] Figure 3 To locate the gene of d2308 dwarf mutant ( Figure 3 A is the gene localization analysis of the d2308 mutant; Figure 3 B is the analysis of mutation positions of d2308 and br2 mutants; Figure 3 C is the sequencing analysis of mutation sites of d2308 and br2 mutants);

[0023] Figure 4Allelic verification of dwarf mutant effect gene br2-d2308 ( Figure 4 A is the phenotypic comparison of B73, d2308, d2308×br2 and br2 plants; Figure 4 B is the statistical analysis of plant height; Figure 4 C is the statistical analysis of ear height; Figure 4 D is the statistical analysis of the last node height, including the length of the tassel);

[0024] Figure 5 Sequence analysis for the development of dCAPS markers based on the br2-d2308 gene (in the figure, B73-Ref is the B73 maize reference genome sequence; B73-dCAPS is the amplified sequence for the development of dCAPS markers for B73 maize; d2308-dCAPS is the amplified sequence for the development of dCAPS markers for the d2308 mutant);

[0025] Figure 6 An electrophoresis analysis was developed for the dCAPS marker based on the br2-d2308 gene (M in the figure: DNA Marker 500, the band sizes from top to bottom are 500, 400, 300, 200, 150, 100 and 50 bp, respectively; 1: electrophoresis of B73 amplification products before restriction digestion; 2: electrophoresis of amplification products of the d2308 mutant before restriction digestion; 3: electrophoresis of amplification products of B73 corn after restriction digestion; 4: electrophoresis of amplification products of the d2308 mutant after restriction digestion). DETAILED DESCRIPTION

[0026] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0027] 1. Materials

[0028] 1.1 The maize dwarf mutant d2308 used in this experiment was created by EMS mutagenesis, with the deposit number CCTCCNO: P202426. The br2 mutant is a reported loss-of-function mutant of the Br2 gene (Zhao et al. An EMS-induced allele of the brachytic2 gene can reduce plant height in maize. Plant Cell Rep. 2023, 42(4): 749-761).

[0029] 1.2 The B73 maize inbred line used in this experiment was maintained by the National and Local Joint Engineering Laboratory for Crop Stress Breeding and Disaster Reduction, College of Life Sciences, Anhui Agricultural University.

[0030] All reagents used in this experiment are conventional reagents unless otherwise specified, and are prepared with deionized water. All instruments used are conventional laboratory instruments. All methods used in this experiment are conventional methods known to technicians in this field unless otherwise specified.

[0031] 2. Methods

[0032] 2.1 Creation of maize EMS mutants

[0033] After the female ears of corn inbred line B73 are drawn out, they are bagged with kraft paper bags. At the peak of pollen shedding, fresh pollen is collected and anthers are filtered with a sieve. Ethyl methane sulfonate (EMS) mother solution is added to mineral oil at a ratio of 1:1000 to prepare a working solution. The pollen and EMS working solution are evenly mixed at a ratio of about 1:10 (V / V), and left to stand in the dark for 40 minutes, with a gentle shake every 10 minutes. Use a brush or a small brush to evenly apply the above pollen mixture on the corn silk, and bag the female ears again to mark them. After the ears mature, the M1 generation seeds are harvested, and the M1 seeds are sown individually and self-pollinated to obtain the M2 generation. 20-40 seeds are randomly selected from the M2 generation ears for single-seed single-row sowing and phenotypic screening.

[0034] Among the identified M2 offspring phenotypic variant plants, one plant had a significant decrease in plant height and ear height, good ear setting, and no other obvious adverse traits. After self-pollination, the dwarfed plant was planted in a uniform test field with the B73 corn inbred line, and it was found that it still showed dwarfing traits. After backcrossing the dwarfed plant and self-pollinating for two consecutive generations, the dwarfing phenotype could still be stably inherited, indicating that the dwarfing trait of the plant may be caused by gene mutation caused by EMS mutagenesis, and the dwarfed plant was named dwarf mutant d2308. The dwarf mutant corn seed Zea mays L.d2308 obtained by EMS mutagenesis was registered and deposited in the China Center for Type Culture Collection, with the deposit address at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on October 9, 2024, and the deposit number is CCTCC NO: P202426.

[0035] 2.2 Phenotypic analysis and yield trait analysis of dwarf mutant d2308

[0036] 2.2.1 The dwarf mutant d2308 obtained in step 2.1 above was systematically compared with the phenotype of corn B73. Figure 1As shown in the figure, compared with B73 plants, the plant height and ear height of d2308 decreased by about 31.8% and 51.5%, respectively, and the ear height decreased more. The leaf length of the dwarf mutant d2308 was significantly reduced compared with that of B73 plants, the leaf width was significantly increased, the leaf angle of the second leaf was significantly reduced, and the leaf angle of the first leaf above the ear and the first leaf below the ear increased to a certain extent. At the same time, the stem thickness of the third internode above the ear of d2308 plants increased significantly, indicating that the lodging resistance and density tolerance of the dwarf mutant d2308 may be significantly improved.

[0037] 2.2.2 The yield per plant and other related traits of the B73 inbred line and the dwarf mutant d2308 obtained in step 2.1 above were analyzed to further analyze their potential for increasing plant density and yield. Figure 2 As shown in the figure, compared with the B73 inbred line, the ear length, grain length and grain thickness of d2308 decreased to varying degrees, and the grain width increased significantly, but there was no significant difference in the 100-grain weight and seed yield rate between B73 and d2308. Further analysis of the grain weight per ear of d2308 and B73 showed that the yield loss per plant of d2308 was about 5.7%. Therefore, when the plant height of d2308 was significantly reduced, its yield loss was small, and it has great potential for application in dwarfing breeding to increase density and yield.

[0038] 2.3 Analysis of genetic rules of dwarf mutant d2308

[0039] In order to analyze whether the dwarf mutant d2308 was caused by a single gene mutation, the dwarf mutant d2308 obtained in step 2.1 was hybridized with B73 to obtain F1 plants, and the F2 segregation population was constructed by selfing. The F2 population was sown in a uniform test field, and the segregation ratio was analyzed with plant height as the main trait 10-15 days after pollination. By counting the F2 segregation population in the field, 28 dwarf plants with a phenotype similar to d2308 appeared among 106 plants. The chi-square test (χ 2 =0.11<χ 2 0.05 =3.84), which is consistent with the theoretical segregation ratio of 3:1, indicating that the dwarf phenotype of d2308 is controlled by a recessive single gene.

[0040] 2.4D2308 mutant gene localization

[0041] The plants with d2308 dwarf phenotype in the F2 population in step 2.3 above were sampled individually, and the genomic DNA of 19 individual plants was extracted and quality tested. After the quality inspection was qualified, the extracted genomic DNA of the individual plants was mixed with equal quality and proportion to build a library, and then subjected to BSA high-throughput sequencing. The sequencing data was quality controlled and analyzed, the filtered sequencing reads were aligned and assembled with the maize B73 genome (V4.32), and the mutated SNP sites were analyzed. The optimized Mutmap positioning analysis package was used to screen the SNP sites of the mutation effect gene, and the SNP screening was performed according to the G->A (C->T) base mutation mode and the SNP-Index=1.0 standard, and the variation types of the screened SNPs were analyzed, including premature termination / termination loss of transcription, splicing site donor / acceptor, start codon ATG gain / loss, non-synonymous mutation, etc. At the same time, the correspondence diagram with the chromosome position was drawn according to the average SNP-Index value of the 3Mb interval to screen the main effect SNP sites.

[0042] According to the above analysis method, a significant signal peak was found on the corn No. 1 stain ( Figure 3 A), the SNP sites selected in this interval and their corresponding genes were annotated and analyzed. A SNP site was found in this interval that met the screening criteria of SNP-Index=1.0 and G->A (C->T) base mutation mode, and the SNP site occurred at the 1276bp position of the 5th exon of the Br2 gene (such as Figure 3 B), G mutated to A, resulting in glycine (G) mutated to arginine (R). The Br2 gene has been reported to affect the regulation of plant height development. Combined with the dwarf phenotype of d2308, it is speculated that the mutation at this site may be the d2308 effect mutation site. To further verify the authenticity of the mutation at this site, specific primers were designed around the mutation site, and Primer STAR Max Premix (2×) was used to perform mixed PCR amplification on BSA sequencing DNA samples, and B73 plants were used as controls;

[0043] The primers for PCR amplification are as follows:

[0044] SEQ ID NO.3: d2308-F: 5'-GGGGTTCGTCCTCCAGTG-3';

[0045] SEQ ID NO. 4: d2308-R: 5'-GGCTCCACCTCCGTCTTG-3'.

[0046] The sample loading system for PCR amplification is shown in Table 1:

[0047] Table 1 PCR sample loading system

[0048]

[0049] The reaction procedure of PCR amplification was as follows: denaturation at 98°C for 5 min; denaturation at 98°C for 10 sec, annealing at 59°C for 15 sec, extension at 72°C for 30 sec, 35 cycles; extension at 72°C for 10 min, and storage at 4°C. The fragments amplified by PCR were detected by 2.0% agarose gel electrophoresis and then sequenced. The sequencing results were analyzed using Sequencher software, and it was found that a homozygous mutation from G to A occurred at the predicted mutation position (e.g. Figure 3 C and SEQ ID NO.1-2, the nucleotide sequence of the 5th exon of the Br2 gene of the dwarf mutant d2308 is shown in SEQ ID NO.1, and the nucleotide sequence of the 5th exon of the Br2 gene of the wild type B73 is shown in SEQ ID NO.2), which proves the authenticity of the mutation site.

[0050] 2.5 Allelic verification of dwarf mutant effect gene br2-d2308

[0051] In order to further verify that the mutation occurring in the fifth exon of the Br2 gene is the effect mutation site of the dwarf mutant d2308, another mutant of the Br2 gene (named br2 mutant; Zhao et al. An EMS-induced allele of the brachytic2 gene can reduce plant height in maize. Plant Cell Rep. 2023, 42(4): 749-761) was used to perform an allelic test of gene function. The detection primers are as follows:

[0052] SEQ ID NO.5: br2-F: 5'-CTCATCCGCATGCAGGAG-3';

[0053] SEQ ID NO. 6: br2-R: 5'-CGAACATCTTCTCGCGCA-3'.

[0054] Test results such as Figure 3 As shown in C, the br2 mutant is a mutation from G to A occurring in the fifth exon of the Br2 gene, which causes the translation of the gene to terminate prematurely and the plant exhibits a more significant dwarf trait.

[0055] The mutant d2308 was crossed with the br2 mutant to obtain F1 plants, and then the F1 plants of B73, d2308, br2, and d2308×br2 were planted in a uniform experimental field for phenotypic analysis such as plant height, e.g. Figure 4As shown, among the four different types of germplasms of the Br2 gene, the br2 mutant had the shortest plant height, the d2308 mutant had a higher plant height than br2, but was significantly shorter than the B73 plant, and the plant height phenotype of the F1 generation plants of d2308×br2 was similar to that of d2308, and did not restore the plant height of the B73 plants, indicating that the mutation from G to A in the fifth exon of the Br2 gene was the effect mutation site of d2308.

[0056] 3. Development of dCAPS markers based on br2-d2308 gene

[0057] Leaves of wild-type maize B73 plants and d2308 dwarf mutant plants at the 3-leaf stage were taken and their genomic DNA was extracted using the CTAB method. After the quality was qualified by electrophoresis, it was used for subsequent PCR amplification and marker development. Figure 5 As shown, according to the gene localization results of the d2308 dwarf mutant, dCAPS marker development was performed on the d2308 mutation site using dCAPS Finder 2.0 (http: / / helix.wustl.edu / dcaps / dcaps.html). 25bp sequences were taken from both ends of the d2308 mutation site and the site corresponding to B73, and entered into the corresponding text boxes of the dCAPS Finder 2.0 website. According to the sequence analysis results and the characteristics of the endonuclease, the number of primer mismatches was set to 2 bases, and the endonuclease was selected as ApaI (GGGCCC) for specific primer design. The length of the amplified sequence was 211bp, and the wild-type corn B73 sequence after amplification could be cut into (25bp+186bp). The amplification primer sequence is as follows:

[0058] SEQ ID NO.7: M2308-F: 5'-TGCTGCAGAAGATGTTCATGAAGG-3';

[0059] SEQ ID NO.8: M2308-R: 5'-CCGTAGCCGCTGCCGGCGAT GG GCC-3' (mismatched bases are underlined).

[0060] The B73 and d2308 genomes were amplified using 2×Accurate Taq Master Mix (dye plus), and the PCR amplification sample addition system was referred to Table 1. The PCR amplification reaction program was as follows: 94°C pre-denaturation for 5 min, 98°C denaturation for 10 sec, 67°C annealing for 30 sec, 72°C extension for 20 sec, 35 cycles, and 72°C total extension for 10 min. After the PCR reaction was completed, 3% agarose gel electrophoresis was used for detection, and it was found that the product size was consistent with the theoretical fragment size, and the target band was cut and recovered for later use.

[0061] The purified PCR products of B73 and d2308 were ligated to blunt T vectors respectively. The ligation systems are shown in Table 2:

[0062] Table 2T vector connection and sample loading system

[0063]

[0064] After mixing, place in a 25°C metal bath for 30 minutes to complete the connection. The connection product is transformed into Escherichia coli DH5α competent cells by heat shock method. After picking single clone colonies for culture, they are sent to a biological company for sequencing for verification.

[0065] The purified PCR products of B73 and d2308 were digested with ApaI, respectively, and the loading system was shown in Table 3:

[0066] Table 3 Enzyme digestion and sample addition system

[0067]

[0068] After mixing, place in a 37°C metal bath for 30 minutes, and use the PCR products of B73 and d2308 without enzyme digestion as controls for detection using 3% agarose gel electrophoresis. Figure 6 As shown in the figure, the electrophoresis results showed that the migration speed of the electrophoresis band of the PCR product amplified using B73 genome DNA as a template after enzyme digestion (lane 3) was significantly faster than that of d2308 (lane 4), while there was no significant difference between the two PCR products before enzyme digestion, indicating that the dCAPS marker was successfully developed and can be used in marker-assisted selection in dwarfing improvement breeding.

[0069] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A corn dwarfing gene br2-d2308, characterized in that: The br2-d2308 gene is obtained by mutating the 1276th base of the 5th exon of the corn Br2 gene from G to A. The nucleotide sequence of the 5th exon of the br2-d2308 gene is shown in SEQ ID NO.

1.

2. An application of the maize dwarfing gene br2-d2308 as claimed in claim 1 in maize molecular breeding.

3. The use according to claim 2, characterized in that: The corn germplasm to be improved is hybridized and backcrossed with the corn germplasm containing the corn dwarfing gene br2-d2308, and the offspring containing the relevant traits of the corn germplasm to be improved and the corn dwarfing gene br2-d2308 are screened to obtain new dwarfed corn germplasm.

4. The use according to claim 2, characterized in that: By using gene editing technology, the 1276th base of the 5th exon of the Br2 gene in the genome of the corn to be improved was mutated from G to A, thus obtaining a dwarf strain of the corn germplasm to be improved.

5. A molecular marker based on the maize dwarfing gene br2-d2308, characterized in that: The molecular marker is a dCAPS marker, which includes an amplification primer for amplifying a nucleotide sequence containing the 1276th base of the 5th exon of the maize Br2 gene, and the base type of the 1276th base is G / A; Also included are restriction enzymes for base typing verification.

6. The molecular marker according to claim 5, characterized in that The restriction endonuclease is ApaI.

7. The molecular marker according to claim 5, characterized in that The nucleotide sequence of the amplification primer is: SEQ ID NO.7: M2308-F: 5'-TGCTGCAGAAGATGTTCATGAAGG-3'; SEQ ID NO. 8: M2308-R: 5'-CCGTAGCCCGCTGCCGGCGATGGGCC-3'.

8. Use of the molecular marker according to any one of claims 5 to 7 in assisting the breeding of dwarf corn varieties.

9. The use according to claim 8, characterized in that: The genomic DNA of the tested corn germplasm was used as a template, and the target sequence was amplified by PCR using the amplification primers labeled with dCAPS. The PCR amplification product was digested with the restriction endonuclease ApaI, and the genotyping of the digestion product was performed to obtain the molecular marker type of the tested corn germplasm. The plant height of the tested corn germplasm or variety with the molecular marker type of A is significantly shorter than the plant height of the tested corn with the molecular marker type of G.

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