CN114774568B - Molecular marker of maize half-dwarf gene ZmD13 and application thereof - Google Patents

Abstract

The invention discloses a molecular marker of a maize half-dwarf gene ZmD13 and application thereof. One technical scheme to be protected by the invention is a primer composition for detecting polymorphism or genotype of SNP1477 in corn genome, wherein the primer composition consists of two single-stranded DNA shown as sequence 2 and sequence 3 in a sequence table; the SNP1477 is a SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table. The corn SNP1477 locus dCAPS marker developed by the invention has an important effect on auxiliary locus selection and can be used for identifying whether corn contains dwarf genes. The dCAPS marker can accurately, quickly and efficiently identify the mutation site genotype of the dwarf gene d13, and can be applied to corn plant height breeding and improvement.

Description

Molecular marker of maize half-dwarf gene ZmD13 and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a molecular marker of a maize semi-dwarf gene ZmD and application thereof.

Background

Corn is an important food crop, as well as an important animal feed and industrial feedstock. In the past century, the increase in yield per unit area of corn was not only dependent on the increase in yield per plant, but also an increase in planting density was an important factor. The corn density-resistant variety has the characteristics of proper plant height, compact plant type, developed root system, tough stalks, stress resistance, disease resistance and the like. Plant height is one of the key traits affecting the confidentiality of maize varieties. Reasonably reducing the plant height of the variety is beneficial to enhancing the lodging resistance of corn, improving the planting density, improving the population permeability and optimizing the light energy utilization efficiency, thereby realizing the aim of improving the yield per unit area. Therefore, the method for excavating the plant height regulating gene and breeding the excellent dwarf variety is an important direction of corn density-resistant breeding.

The development of rice and wheat dwarf gene rht1 and sd1 and the popularization and application of related dwarf derived line varieties in the 60 s of the last century have improved a green revolution, and the yield of wheat and rice is greatly improved worldwide. In corn, the plant height regulating genes are discovered and relevant functional markers are developed, so that the method has important significance for breeding and application of short-stalk corn varieties.

SNP markers are usually verified by the Sanger sequencing method, but at a higher price and with a lower throughput. If SNP is converted into CAPS or dCAPS polymorphism co-dominant markers which can be verified by enzyme digestion, SNP locus detection can be efficiently and rapidly realized by simple PCR amplification, restriction enzyme digestion and product gel electrophoresis. Because of these advantages, CAPS and dCAPs labeling techniques have become important molecular marker techniques in biological research, and are widely used in the fields of genetic map construction, germplasm identification, assisted breeding, and the like.

Disclosure of Invention

The invention aims to solve the technical problem of identifying whether plants contain dwarf genes.

In order to solve the above technical problems, the present invention provides a primer composition for detecting polymorphism or genotype of SNP1477 in corn genome. The primer composition consists of two single-stranded DNAs shown as a sequence 5 and a sequence 6 in a sequence table.

The SNP1477 is a SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table.

In the primer composition described above, the molar ratio of the two single-stranded DNA may be the same.

In order to solve the technical problems, the invention also provides a product for detecting polymorphism or genotype of SNP1477 locus in corn genome. The product contains the primer composition described above. The SNP1477 locus is one SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table.

The product described above may be a reagent or a kit.

In order to solve the above technical problems, the present invention also provides a method for preparing the primer composition described above. The method may comprise the step of packaging the two single stranded DNA fragments described above separately.

In order to solve the technical problems, the invention also provides a method for detecting polymorphism or genotype of SNP1477 locus in corn genome. The method may comprise the steps of:

1) PCR amplification was performed using the genomic DNA of maize as a template and the primer composition described above to obtain a PCR amplification product.

2) And (3) carrying out enzyme digestion on the PCR amplification product obtained in the step (1) by using restriction enzyme TaqI to obtain an enzyme digestion product, and determining the polymorphism or genotype of the SNP1477 locus according to the size of the enzyme digestion product.

The SNP1477 locus is one SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table.

The corn enzyme digestion product with the genotype of CC at the SNP1477 locus can be two bands (185 bp and 23 bp), and the nucleotide sequences of the two bands can be respectively shown as a sequence 8 in a sequence table and a sequence 7 in the sequence table. The corn enzyme digestion product with the genotype of CT at the SNP1477 locus can be three bands (185 bp, 23bp and 208 bp), and the nucleotide sequences of the three bands can be respectively shown as a sequence 8 in a sequence table, a sequence 7 in the sequence table and a sequence 9 in the sequence table. The corn enzyme digestion product with the genotype TT at the SNP1477 locus can be a band (208 bp), and the nucleotide sequence of the band can be respectively shown as a sequence 9 in a sequence table.

Any of the following applications of the primer composition described above is also within the scope of the present invention:

a1 Use of the primer composition described above for detecting polymorphism or genotype at SNP1477 site in corn genome.

A2 The application of the primer composition in detection of dwarf corn.

A3 The use of the primer composition described above in maize breeding and improvement.

The SNP1477 locus is one SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table.

In order to solve the above technical problem, the present invention also provides any one of the following applications of the product described above:

b1 Use of the above-described product for detecting a polymorphism or genotype at SNP1477 site in corn genome;

b2 The application of the product in detecting dwarf corns.

B3 The use of the above-described products in corn breeding and improvement.

The SNP1477 locus is one SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table.

Any of the following applications of the method described above is also within the scope of the present invention:

c1 Use of the method described above for detecting polymorphism or genotype at SNP1477 site in maize genome.

C2 Use of the method described above for detecting dwarf maize.

C3 Use of the methods described above in corn breeding and improvement.

The SNP1477 locus is one SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table.

The maize may be selected from the progeny of crosses of maize dwarf mutant d13×maize inbred line B73, such as the F2 generation or more; the maize dwarf mutant d13×inbred line Chang 7-2 may also be selected from the filial generation such as F2 generation or more.

The invention discovers a recessive single-gene controlled semi-dwarf mutant dwarf13 (d 13) in a corn EMS mutant library, then clones a novel gene ZmD which participates in the corn plant height development regulation, and discovers that the SNP1477 locus of the ZmD gene has the nucleotide type of C or T, which is a key locus for generating a d13 dwarf phenotype; on the basis, the development of the corn SNP1477 locus dCAPS mark has an important role in auxiliary locus selection, and can be used for identifying whether corn contains dwarf genes. The dCAPS marker can accurately, quickly and efficiently identify the mutation site genotype of the dwarf gene d13 so as to assist the application of the site in corn plant height breeding and improvement.

Drawings

FIG. 1 shows the phenotype analysis of mutant d13 and Wild Type (WT). (A-D): mutant d13 was compared to WT phenotype; (E): d13 vs WT internode length; (F): d13 represents P <0.01 compared to WT phenotype values.

FIG. 2 is a CRISPR/Cas9 editing mutation of the ZmD13 gene. (a) editing the mutant in comparison to a wild-type (WT) phenotype; (B) editing target sequencing analysis; (C) Editing mutant compared with Wild Type (WT) strain height and spike height values, representing P <0.01; (D) Editing mutants were compared to Wild Type (WT) internode length.

FIG. 3 is F ₂ Population construction process.

FIG. 4 is a comparison of d13 with wild type phenotype. The short stem mutant d13 has a height of about 50% of that of the wild type, the stem nodes are obviously shortened, and the leaf length is shorter than that of the wild type.

Fig. 5 shows dCAPS marker development.

FIG. 6 Wild Type (WT), d13 mutant and F ₁ dCAPs primer amplification and cleavage.

FIG. 7 is F ₂ Enzyme-cleaved bands of the population.

FIG. 8 shows the enzyme-digested strips of mutants and breeding inbred lines.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Sources of corn materials used in the examples of the present invention:

f is obtained by hybridization construction of mutant d13 and maize B73 inbred line ₂ An isolated population (fig. 3);

the 20 breeding inbred lines Zheng 58, chang 7-2, mo17, B73, heddle 31, zizania 319, huangzao four, huangC, 178, ye 478, dan 340, shen 330, shen 137, PH4CV, PH6WC, CL11, NG5, HD568, 11DM124 and B195 are stored in the laboratory, and related documents: li Yu, wang Tianyu. Formation of maize-breeding germplasm basis and diaphysis parent in China maize science 2010, 18 (5): 1 to 8; hu X, wang H, et al Transcriptome profiling and comparison of maize ear heterosis during the spikelet and floret differentiation stages.BMC genomics.2016Nov 22;17 (1) 959.Doi:10.1186/s12864-016-3296-8.PMID:27875998.

Example one discovery and cloning of novel Gene ZmD13 for corn plant height development control

1. Screening and phenotype identification of corn plant height mutant d13

1.1 screening of maize Strain height mutant d13

EMS mutagenesis is carried out on the maize inbred line Chang 7-2 material, and about 10,000 pieces of mutagenesis generation (M) is obtained after the mature of the EMS mutagenesis clusters ₁ ) Seed, M ₁ The seeds are sown singly and harvested singly after selfing. M is M ₂ And carrying out trait investigation from seedling stage to mature stage by using 20 seed lines of the generation seeds. In one of the spike lines, 5 half dwarf mutants were found, designated dwarf13 (d 13). Then selfing the d13 mutant spike to obtain M ₃ And M ₄ The post-representation observation shows that the semi-dwarf mutation character can be inherited stably.

The dwarf phenotype of the corn d13 mutant can be observed in the 5-leaf stage, and the jointing stage is more obvious. Mature d13 plant height and ear position are only about 50% of wild-type Chang 7-2 (represented by WT in FIG. 1) (A in FIG. 1). Further investigation of the number and length of the stem nodes of the mutant and wild type revealed that the d13 stem node number was not significantly different from that of the wild type WT, but each stem node was shorter in length than the wild type (B and E in fig. 1). Of these, 3-node on the ear and 4-node below the ear are shortened most significantly. For a pair ofM ₄ Other agronomic traits of wild-type WT and d13 mutants in the generation population were investigated and found that in addition to dwarfing, the leaf and tassel spindle lengths of the d13 mutant were shortened (C, D and F in fig. 1).

1.2 genetic analysis and map-based cloning of the ZmD13 Gene

Construction of F by crossing d13 (male parent) with B73 (female parent) inbred line ₂ The segregating population, phenotypic investigation and chi-square test showed that the segregation ratio of dwarf to normal plants was consistent with 3:1, d13 mutation character is controlled by recessive single gene.

Extracting parents and F ₂ DNA of 78 d13 mutants in the population was isolated and the genotype of the sample was detected using a targeted sequencing genotyping technique (20K GBTS). Through data analysis, 7,029 polymorphic SNP markers which are available among parents uniformly cover 10 chromosomes of corn, and the average SNP of every Mb is about 3. And respectively calculating SNP index (namely genotype frequency of mutant parent d 13) of each SNP marker, and analyzing the change rule of the SNP index of the whole genome. Within the short arm 1-18Mb region of chromosome 3, the marker SNP index value exceeds 0.5 and gradually approaches 1, indicating that the region is linked with the target trait and is a candidate segment. Genotypic variation of 78 samples in this section was analyzed and found that the minimum initial localization interval was located between 4.1 and 6.0 Mb. Further expanding the positioning population, developing 7 InDel markers with polymorphism between parents, and reducing the interval to 200kb. According to the predicted result of Gramen (http:// Gramen. Org /) website, there are 5 protein coding genes in this interval. Sequencing analysis shows that one new gene encoding cytochrome P450 enzyme has one C-T mutation in the 8 th exon and accords with the EMS mutation characteristic. The single base variation forms a stop codon, the d13 mutant protein translation is terminated in advance, and compared with the wild type, the gene is predicted to be a candidate gene by lacking 10 amino acids, and the candidate gene is named ZmD13.

The CDS sequence of the ZmD gene is shown as a sequence 1 in a sequence table, and y is c or t in the sequence 1. The coding sequence of the d13 mutant protein is shown as a sequence 2 in a sequence table.

1.3 CRISPR/Cas9 knockout validation of ZmD13 gene

2 targets were selected on the active center heme binding domain (heme binding domain) sequence of the ZmD protein using CRISPR/Cas9 base vector (CPB vector) (China academy of agricultural sciences crop science research Xie Chuanxiao researchers benefit related literature: li C, et al, RNA-guided Cas9 as an in vivo desired-target mutator in mail.plant Biotechnol J.2017Dec;15 (12): 1566-1576.Doi:10.1111/pbi.12739.Epub 2017May 12.PMID:28379609). And constructing an expression cassette of the double-target sgRNA and the Cas9 protein, and inserting the expression cassette into a binary expression vector CPB to finish CRISPR/Cas9 knockout vector construction.

The two ZmD targeted sgrnas were sgRNA1 and sgRNA2, respectively. The nucleotide sequence of the sgRNA1 target is 5'-GAAGAGGCGGAAAACCGACG-3', and the 1044 th to 1063 rd positions of the sequence 2 in the sequence table of the ZmD gene are targeted; the nucleotide sequence of the sgRNA2 target is 5'-GGCTTCACCCCTTTTGGCGG-3', and the 1324 th-1343 rd site of the sequence 1 in the sequence table of the ZmD gene is targeted.

Obtaining and purifying the fragment of interest (expression cassette of double-target sgRNA and Cas9 protein):

primers used for preparing target fragment:

TABLE 1 primer sequences

The target fragment U6-2 promoter is obtained by amplifying site1-U6-2-F (site 2-U6-2-F) and site1-U6-2-R (site 2-U6-2-R) primers. The target fragment sgRNA fragment is obtained by amplifying site1-sgRNA-F (site 2-sgRNA-F) and site1-sgRNA-R (site 2-sgRNA-R) primers. The method comprises the following specific steps: taking CPB carrier as DNA template, carrying out PCR reaction, and carrying out pre-denaturation for 3min at 95 ℃ in an amplification procedure; denaturation at 95℃for 30s, annealing at 58℃for 20s, elongation at 68℃for 20s,35 cycles; extending at 68deg.C for 5min, and preserving at 12deg.C.

After detection by 1% agarose gel electrophoresis, the U6-2 promoter and sgRNA fragments were gel recovered using the M5 Gel Extraction Kit kit.

The preparation of the target fragment mainly uses an overlap PCR technology, and the overlap PCR reaction is carried out to obtain the overlap fragment, wherein the overlap PCR product contains a target fragment, namely U6-2:: gRNA:: sgRNA (U6-2:: gRNA1:: sgRNA or U6-2:: gRNA2:: sgRNA) (300-802 nucleotide of a sequence 3 in a sequence table or 803-1305 nucleotide of the sequence 3 in the sequence table).

Preparation and recovery of CPB cleavage vector: CPB vector was digested with HindIII and the reaction system was 50. Mu.L: 5. Mu.L of CPB vector DNA (200 ng/. Mu.L), 5. Mu.L of 10 XBuffer, 1. Mu.L of HindIII endonuclease, 39. Mu.L of ddH ₂ O。

Ligation and transformation of target fragment and enzyme-cleaved vector

Using HieffPlus One Step Cloning Kit A kit (Shanghai, hitachi, st. Biotechnology Co., ltd., cat#10911es 20) was ligated with the linear vector (i.e., CPB digestion vector described above) and the target fragment (i.e., overlapping PCR product described above) to obtain a recombinant vector, which was transformed into DH 5. Alpha. Competent cells, followed by colony PCR and bacterial sample sequencing (primers: 5'-AACTGTAGAGTCCTGTTGTC-3' and 5'-CATTCGCCATTCAGGCTGC-3'), and the recombinant vector positive clone was found to contain a double-target U6-2:: gRNA1:: sgRNA::: U6-2: gRNA2: sgRNA (sequence 3 in the sequence table) and an expression cassette (sequence 4 in the sequence table) for Cas9 protein. Shaking culture of positive monoclonal bacteria, extracting CRISPR-Cas9 recombinant plasmid ZmD-gRNA 1-gRNA2, transforming agrobacterium EHA105 strain to obtain recombinant agrobacterium EHA 105/ZmD-gRNA 1-gRNA2, transforming maize B104 (Beijing Bomeixing Ortho technology Co., ltd.) young embryo with recombinant agrobacterium EHA 105/ZmD-gRNA 1-gRNA2 to obtain gene editing maize T ₀ Plants of the generation T ₀ Selfing of maize to obtain T ₁ And (3) replacing.

Editing T for Gene ₀ And T ₁ The target sequence of the generation plant is subjected to PCR amplification detection and sequencing by using primers 5'-GCGCCTAAACATCAGCAGA-3' and 5'-ACAACACCGAGGAGCACAC-3', so that 2 double homozygous mutants are obtained and are respectively named ZmD13-cr1 and ZmD13-cr2; for 2 pairs ofThe phenotype of the homozygous mutants was determined and observed, and the results showed that the phenotype of 2 double homozygous mutants was highly similar to that of the d13 mutant, and that the phenotype was manifested by shortened stem node, stunted plant, and about 61.9% -69.5% and 90.3% -92.9% of the wild type transgenic receptor inbred line B104 (WT in FIG. 2), respectively (Table 2 and FIG. 2). According to the results, zmD13 has the function of regulating and controlling the plant height of corn.

TABLE 2 comparison of height and ear position of Gene editing mutant and wild type B104 plant

Example two, development and application of dCAPS molecular marker of maize half-dwarf Gene ZmD13

The eighth exon of the maize cytochrome P450 enzyme gene ZmD gene, namely the 1477 nucleotide site of the sequence 1, is a SNP site, which is named SNP1477, and the nucleotide of the SNP1477 site is C or T. The nucleotide of SNP1477 site of ZmD gene in corn semi-dwarf mutant dwarf13 (d 13) is T, which is a key site for generating d13 dwarf phenotype. In the field identification of plant height phenotype in the later period of corn plant powder scattering, the short stem mutant d13 has the plant height of about 50% of wild type, each stem node is obviously shortened, and the leaf length is shorter than that of the wild type (figure 4).

Development of dCAPS markers

dCAPS primer development and endonuclease selection were performed on the SNP1477 site of the ZmD gene by using a dCAPS Finder 2.0Program (http:// helix. Wust. Edu/dCAPS. Html), and a target fragment containing the SNP1477 mutation site was amplified, with the amplified fragment length of 208bp. The corresponding downstream primers were designed using Primer Premier 5.0 software and Blast verified for Primer specificity in NCBI. Primers were synthesized by the division of biological engineering (Shanghai) and the specific sequences are shown in Table 3:

TABLE 3 dCAPS primer sequences

dCAPS primer specificity and detection of fragment polymorphisms

The nucleotide at SNP1477 site in maize homozygous wild type such as maize B73 inbred line and wild type inbred line Chang 7-2 is C. The maize d13 dwarf mutant is a homozygous mutant, and the nucleotide at the SNP1477 site of d13 is T. Using dCAPS technology, a mutant base T was introduced 2bp upstream of this SNP, thereby introducing 1 TaqI cleavage site at the wild-type maize SNP1477 site (WT stands for wild-type in FIG. 5). The dCAPS primer amplified product of the wild SNP1477 locus theoretically comprises 1 TaqI restriction enzyme cleavage site recognition sequence, and can be cut into two fragments of 23bp (sequence 7 in a sequence table) and 185bp (sequence 8 in the sequence table) by TaqI restriction enzyme; dCAPS amplified product of SNP1477 site of mutant d13 has no restriction enzyme site, and the length of the band is 208bp (sequence 9 in the sequence table).

Using the dCAPS primer pair designed in step 1, short-stalk mutant d13, wild-type inbred line Chang 7-2 (WT) and F thereof ₁ The hybrid was PCR amplified to give three PCR products, and 1% agarose gel electrophoresis was used to amplify a single band (208 bp each), which was clear and bright, and the size was correct (bands in the PCR product lanes in FIG. 6). The three PCR amplified products were digested with TaqI restriction enzyme, reacted at 65℃for 10 minutes, and then electrophoresed on 3% agarose gel. The results of the experiment show that the three PCR products are cut by enzyme and have 3 types of bands, namely A (208 bp), B (185+23 bp) and H (208+185+23 bp), but the band of 23bp is basically invisible on the gel due to too short. The d13 mutant band was A-type band (d 13 in TaqI cleavage lane in FIG. 6), the Chang 7-2 band was B-type band (WT in TaqI cleavage lane in FIG. 6), F ₁ The hybrid band was H-shaped (F in TaqI cleavage lane in FIG. 6) ₁ ). The dCAPS marker has strong specificity and is separated from the character co-dominant, and can be used for detecting mutation sites in d13 mutants, namely SNP1477 site polymorphism.

The experimental procedure was as follows:

corn material DNA extraction:

plant DNA extraction kit (DP 305) from the company of the root biotechnology (beijing) was used.

PCR amplification system and reaction conditions:

amplification system (enzyme used for PCR amplification is Beijing polymerase Biotechnology Co., ltd., MF-002)

Template DNA* <1μg

2×M5 Taq HiFi PCR Mix 10μL

dCAPS1477-F(10μM) 0.5μL

dCAPS 477-R(10μM) 0.5μL

ddH ₂ O is added to 20 mu L

Reaction conditions:

amplification product cleavage system:

TaqI restriction enzyme (ThermoFisher Scientific, FD 0674), recognition site 5'… T-CGA …';

and (3) enzyme cutting system:

PCR product < 1. Mu.g

10x Buffer 2μL

TaqI 1μL

ddH2O is complemented to 20 mu L

And (3) enzyme cutting: preserving at 65deg.C for 10min and 4deg.C.

Gel electrophoresis detection enzyme section:

3% agarose gel, voltage 160v, electrophoresis 40 min.

dCAPS primer labeling at F ₂ Detection in isolated populations

24 parts of F obtained by hybridization construction of mutant d13 and maize B73 inbred line by dCAPS primer marker of SNP1477 site ₂ The isolated population (FIG. 3) was subjected to genotype detection at SNP1477 locus in the same manner as in step 2. As can be seen from the results of FIG. 7, at F ₂ Of the 17 parts of the population of material with a plant height similar to that of the normal maize B73 wild-type material, 6 parts of the detection band type was B (185+23 bp) (F in FIG. 7 ₂ -3、F ₂ -6、F ₂ -10、F ₂ -12、F ₂ -21、F ₂ -24, indicating the 6 parts F ₂ The material is SNP1477The genotype of the spot was homozygous wild type (AA) for CC, and the 11 material detection bands were H (208+185+23 bp) (F in FIG. 7 ₂ -1、F ₂ -4、F ₂ -5、F ₂ -7、F ₂ -8、F ₂ -9、F ₂ -15、F ₂ -17、F ₂ -18、F ₂ -20、F ₂ -22), indicating the 11 parts F ₂ The material is heterozygous (Aa) with genotype CT of SNP1477 locus; another 7 parts of the short stalk mutant material were all of type A (208 bp) after cleavage (F in FIG. 7 ₂ -2、F ₂ -11、F ₂ -13、F ₂ -14、F ₂ -16、F ₂ -19、F ₂ -23) indicating the 7 parts F ₂ The material is a homozygous mutant (aa) with genotype TT at SNP1477 site. For 24 parts of F ₂ The plant height was measured and calculated (table 4) to give an average plant height of homozygous wild type (CC) of: 253.5; the average plant height of heterozygous wild (CT) is 258.0; the average plant height of the homozygous mutant (TT) was 166.6 cm, only 65.7% and 64.6% of the homozygous and heterozygous wild type, and the differences were very significant (t-test, p)<0.01). Therefore, the accuracy of the genotype detection result and the phenotype of the dCAPS primer molecular markers reaches 100.0 percent. F (F) ₂ Genotype distribution in the segregating population met 1:2:1 (χ ² <3.84 And the method accords with the Mendelian genetic rule, and the fact that the maize dwarf trait is controlled by a recessive single gene is verified again.

Watch 4.F ₂ Plant height of colony and genotype thereof

Detection of dCAPS primer markers in maize backbone inbred lines

And (2) detecting backbone inbred lines in 20 corn breeding by further utilizing dCAPS primer of SNP1477 locus, wherein the detection method is the same as that of the step (2). The detection results show that the banding patterns of the products after enzyme digestion are all A type, namely the genotypes of SNP1477 loci of 20 corn inbred lines detected are all homozygous wild type (AA), which indicates that none of backbone inbred lines contain mutant SNP loci (FIG. 8). Therefore, the d13 dwarf mutant can be used for carrying out plant height genetic improvement on corn to create dwarf germplasm, and the development of dCAPS markers at SNP1477 sites plays an important role in auxiliary site selection. The dCAPS marker can accurately, quickly and efficiently identify the mutation site genotype of the dwarf gene d13 so as to assist the application of the site in corn plant height breeding and improvement.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> institute of crop science at national academy of agricultural sciences

<120> molecular marker of maize semi-dwarf gene ZmD13 and application thereof

<130> GNCSQ213712

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 1512

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atgtcgacca ccaccctgca gctggttccg tggccgccgg agccggcgcg cgccgccgtg 60

ctgcttgttg cggcggtcgt ctgcctgtgg gtgcttctgt cacgcaggcg ggccgccgga 120

gggagcaagg acaaggagcg ggcggcccgg ctcccgcccg gcagcttcgg gtggccgctg 180

gtgggcgaga cgctggactt cgtgtcctgc gcctactcct cccgcccgga ggccttcgtc 240

gacaagcgcc gcctgctgca cgggagcgcg gtgttccggt cgcacctgtt cggctcggcg 300

acggtggtga cgtcggacgc ggaggtgagc cggttcgtgc tgcacagcga cgcgcgcgcc 360

ttcgtgccct ggtacccgcg gtcgctgacg gagctcatgg gcgagtcctc catcctgctc 420

atcaacggca gcctgcagcg gcgcgtgcac ggcctcgtcg gcgccttctt caagtcgccg 480

cagctcaagg cgcaggtcac cgccgacatg cagcgccgcc tcgcgcccgc gctcgccgcc 540

tggaaggttc ggtgcgcctc cgcgccgccg ctccgcatcc aggaccacgc caagacgatc 600

gtgttcgaga tcctggtgag gggtctgatc gggctggagg caggcccgga gatgcagcag 660

ctcaagcacc agttccagga attcattgtc ggcctcatgt ccctccccat caagctgccg 720

gggactaggc tctacaggtc cctccaggcc aagaagagga tggccacgct gatacagggg 780

atcatacagg agaagaggcg gcggcggagg gccgcccttg aggatggcgg agagggcgag 840

ggcgaggccg gtcccccgcg cgacgtcatc gacgtgctca taagcggcgg cgacgagctc 900

accgacgagc tcatatccga caacatgatc gacctgatga tccccgccga ggactctgtg 960

cccgtgctca tcacgctcgc cgtcaagtat ctcagcgagt gcccgcttgc tctgcaacaa 1020

cttgaggagg agaacatgca gctgaagagg cggaaaaccg acgtgggaga gaccttgcag 1080

tggacggact acatgtcact gtcgttcaca caacatgtga taacggagac actgcggatg 1140

gggaatatca tcaacgggat catgcgcaaa gcggtgcggg acgtggaggt gaaggggcac 1200

ctcatcccca agggctggtg cgtgttcgtg tacttccggt cggtccacct cgacgacaag 1260

cgctacgacg agccctacag gttcaacccg tggaggtgga aggagaagga cacgagcacc 1320

atgggcttca ccccttttgg cggtgggcag aggctgtgcc caggcctgga tctggccagg 1380

ctggaagctt ccatctttct ccatcacctg gtgaccagct tcaggtgggt ggcggaggag 1440

gaccacatcg tcaacttccc aaccgtgcgg ctcaagygag gcatgcccgt caggctcacc 1500

agcaaagact ag 1512

<210> 2

<211> 1512

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atgtcgacca ccaccctgca gctggttccg tggccgccgg agccggcgcg cgccgccgtg 60

ctgcttgttg cggcggtcgt ctgcctgtgg gtgcttctgt cacgcaggcg ggccgccgga 120

gggagcaagg acaaggagcg ggcggcccgg ctcccgcccg gcagcttcgg gtggccgctg 180

gtgggcgaga cgctggactt cgtgtcctgc gcctactcct cccgcccgga ggccttcgtc 240

gacaagcgcc gcctgctgca cgggagcgcg gtgttccggt cgcacctgtt cggctcggcg 300

acggtggtga cgtcggacgc ggaggtgagc cggttcgtgc tgcacagcga cgcgcgcgcc 360

ttcgtgccct ggtacccgcg gtcgctgacg gagctcatgg gcgagtcctc catcctgctc 420

atcaacggca gcctgcagcg gcgcgtgcac ggcctcgtcg gcgccttctt caagtcgccg 480

cagctcaagg cgcaggtcac cgccgacatg cagcgccgcc tcgcgcccgc gctcgccgcc 540

tggaaggttc ggtgcgcctc cgcgccgccg ctccgcatcc aggaccacgc caagacgatc 600

gtgttcgaga tcctggtgag gggtctgatc gggctggagg caggcccgga gatgcagcag 660

ctcaagcacc agttccagga attcattgtc ggcctcatgt ccctccccat caagctgccg 720

gggactaggc tctacaggtc cctccaggcc aagaagagga tggccacgct gatacagggg 780

atcatacagg agaagaggcg gcggcggagg gccgcccttg aggatggcgg agagggcgag 840

ggcgaggccg gtcccccgcg cgacgtcatc gacgtgctca taagcggcgg cgacgagctc 900

accgacgagc tcatatccga caacatgatc gacctgatga tccccgccga ggactctgtg 960

cccgtgctca tcacgctcgc cgtcaagtat ctcagcgagt gcccgcttgc tctgcaacaa 1020

cttgaggagg agaacatgca gctgaagagg cggaaaaccg acgtgggaga gaccttgcag 1080

tggacggact acatgtcact gtcgttcaca caacatgtga taacggagac actgcggatg 1140

gggaatatca tcaacgggat catgcgcaaa gcggtgcggg acgtggaggt gaaggggcac 1200

ctcatcccca agggctggtg cgtgttcgtg tacttccggt cggtccacct cgacgacaag 1260

cgctacgacg agccctacag gttcaacccg tggaggtgga aggagaagga cacgagcacc 1320

atgggcttca ccccttttgg cggtgggcag aggctgtgcc caggcctgga tctggccagg 1380

ctggaagctt ccatctttct ccatcacctg gtgaccagct tcaggtgggt ggcggaggag 1440

gaccacatcg tcaacttccc aaccgtgcgg ctcaagtgag gcatgcccgt caggctcacc 1500

agcaaagact ag 1512

<210> 3

<211> 1486

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

aggagaacac atgcacacta aaaagataaa actgtagagt cctgttgtca aaatactcaa 60

ttgtccttta gaccatgtct aactgttcat ttatatgatt ctctaaaaca ctgatattat 120

tgtagtacta tagattatat tattcgtaga gtaaagttta aatatatgta taaagataga 180

taaactgcac ttcaaacaag tgtgacaaaa aaaatatgtg gtaatttttt ataacttaga 240

catgcaatgc tcattatctc tagagagggg cacgaccggg tcacgctgca ctgcacaagc 300

taattggccc ttacaaaata gctagacgtg caggtggctg gatgtgcgct ccctgaatat 360

caacttgtgt ctcctccgat tcagtccgca gatgaaactt ggtaataact gcagctgatc 420

cgtcgtcatt catgctatgc aggggattcg atcttcagca tgtgcagtgc aggcaacaac 480

aatctacgtt gtctgggctt gcgataggta cacgaccacg agggaaggca acgcgtgatg 540

tatgggccgc gcctaagcat ccagcccacg cgggcgtgcg cgtcgtcgct acggcttgcg 600

ggggaaggga tcaagggacg aaccgagaac tagtaccaga ccggccagcg agcattgcag 660

acaccggctt ataagttcag ctgcgaccac cgctccgaag aggcggaaaa ccgacggttt 720

tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca 780

ccgagtcggt gcttttttta agctaattgg cccttacaaa atagctagac gtgcaggtgg 840

ctggatgtgc gctccctgaa tatcaacttg tgtctcctcc gattcagtcc gcagatgaaa 900

cttggtaata actgcagctg atccgtcgtc attcatgcta tgcaggggat tcgatcttca 960

gcatgtgcag tgcaggcaac aacaatctac gttgtctggg cttgcgatag gtacacgacc 1020

acgagggaag gcaacgcgtg atgtatgggc cgcgcctaag catccagccc acgcgggcgt 1080

gcgcgtcgtc gctacggctt gcgggggaag ggatcaaggg acgaaccgag aactagtacc 1140

agaccggcca gcgagcattg cagacaccgg cttataagtt cagctgcgac caccgctccg 1200

gcttcacccc ttttggcggg ttttagagct agaaatagca agttaaaata aggctagtcc 1260

gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttaagcttgg cactggccgt 1320

cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 1380

acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 1440

acagttgcgc agcctgaatg gcgaatgcta gagcagcttg agcttg 1486

<210> 4

<211> 6271

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

aagcttgtgc agtgcagcgt gacccggtcg tgcccctctc tagagataat gagcattgca 60

tgtctaagtt ataaaaaatt accacatatt ttttttgtca cacttgtttg aagtgcagtt 120

tatctatctt tatacatata tttaaacttt actctacgaa taatataatc tatagtacta 180

caataatatc agtgttttag agaatcatat aaatgaacag ttagacatgg tctaaaggac 240

aattgagtat tttgacaaca ggactctaca gttttatctt tttagtgtgc atgtgttctc 300

cttttttttt gcaaatagct tcacctatat aatacttcat ccattttatt agtacatcca 360

tttagggttt agggttaatg gtttttatag actaattttt ttagtacatc tattttattc 420

tattttagcc tctaaattaa gaaaactaaa actctatttt agttttttta tttaataatt 480

tagatataaa atagaataaa ataaagtgac taaaaattaa acaaataccc tttaagaaat 540

taaaaaaact aaggaaacat ttttcttgtt tcgagtagat aatgccagcc tgttaaacgc 600

cgtcgacgag tctaacggac accaaccagc gaaccagcag cgtcgcgtcg ggccaagcga 660

agcagacggc acggcatctc tgtcgctgcc tctggacccc tctcgagagt tccgctccac 720

cgttggactt gctccgctgt cggcatccag aaattgcgtg gcggagcggc agacgtgagc 780

cggcacggca ggcggcctcc tcctcctctc acggcaccgg cagctacggg ggattccttt 840

cccaccgctc cttcgctttc ccttcctcgc ccgccgtaat aaatagacac cccctccaca 900

ccctctttcc ccaacctcgt gttgttcgga gcgcacacac acacaaccag atctccccca 960

aatccacccg tcggcacctc cgcttcaagg tacgccgctc gtcctccccc cccccccctc 1020

tctaccttct ctagatcggc gttccggtcc atggttaggg cccggtagtt ctacttctgt 1080

tcatgtttgt gttagatccg tgtttgtgtt agatccgtgc tgctagcgtt cgtacacgga 1140

tgcgacctgt acgtcagaca cgttctgatt gctaacttgc cagtgtttct ctttggggaa 1200

tcctgggatg gctctagccg ttccgcagac gggatcgatt tcatgatttt ttttgtttcg 1260

ttgcataggg tttggtttgc ccttttcctt tatttcaata tatgccgtgc acttgtttgt 1320

cgggtcatct tttcatgctt ttttttgtct tggttgtgat gatgtggtct ggttgggcgg 1380

tcgttctaga tcggagtaga attctgtttc aaactacctg gtggatttat taattttgga 1440

tctgtatgtg tgtgccatac atattcatag ttacgaattg aagatgatgg atggaaatat 1500

cgatctagga taggtataca tgttgatgcg ggttttactg atgcatatac agagatgctt 1560

tttgttcgct tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc gttctagatc 1620

ggagtagaat actgtttcaa actacctggt gtatttatta attttggaac tgtatgtgtg 1680

tgtcatacat cttcatagtt acgagtttaa gatggatgga aatatcgatc taggataggt 1740

atacatgttg atgtgggttt tactgatgca tatacatgat ggcatatgca gcatctattc 1800

atatgctcta accttgagta cctatctatt ataataaaca agtatgtttt ataattattt 1860

tgatcttgat atacttggat gatggcatat gcagcagcta tatgtggatt tttttagccc 1920

tgccttcata cgctatttat ttgcttggta ctgtttcttt tgtcgatgct caccctgttg 1980

tttggtgtta cttctgcaga tggactataa ggaccacgac ggagactaca aggatcatga 2040

tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat 2100

ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc 2160

tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct 2220

gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag 2280

cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg 2340

gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga 2400

cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg 2460

gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat 2520

ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta 2580

tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa 2640

ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct 2700

gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag 2760

actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa 2820

tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa 2880

cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct 2940

ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa 3000

cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc 3060

ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct 3120

gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag 3180

caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt 3240

catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag 3300

agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca 3360

cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga 3420

caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct 3480

ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc 3540

ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat 3600

gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta 3660

cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag 3720

aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac 3780

caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt 3840

cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca 3900

cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat 3960

tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg 4020

gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag 4080

atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc 4140

cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca 4200

gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg 4260

ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa 4320

gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa 4380

gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa 4440

gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat 4500

cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta 4560

cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga 4620

ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa 4680

ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt 4740

cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag 4800

aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg 4860

cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct 4920

ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt 4980

gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt 5040

gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac 5100

cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt 5160

gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa 5220

gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg 5280

cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga 5340

taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt 5400

gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa 5460

cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga 5520

cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa 5580

gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga 5640

gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat 5700

catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc 5760

ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt 5820

cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa 5880

acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga 5940

gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa 6000

caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct 6060

gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag 6120

gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct 6180

gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa 6240

aaaggccggc caggcaaaaa agaaaaagta a 6271

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ttcccaaccg tgcggctcaa t 21

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ttcagtctgc ctaaatacct ccga 24

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ttcccaaccg tgcggctcaa gcg 23

<210> 8

<211> 185

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

aggcatgccc gtcaggctca ccagcaaaga ctagctagct agtcactacg acctgcttgt 60

gcatgccatg catgcagatt tcagtcacgt catatgcaat atatattgga ccctggaggt 120

tgcacgtgta cagaagagag gatgcaggag ctagcagatg atcggaggta tttaggcaga 180

ctgaa 185

<210> 9

<211> 208

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ttcccaaccg tgcggctcaa gcgaggcatg cccgtcaggc tcaccagcaa agactagcta 60

gctagtcact acgacctgct tgtgcatgcc atgcatgcag atttcagtca cgtcatatgc 120

aatatatatt ggaccctgga ggttgcacgt gtacagaaga gaggatgcag gagctagcag 180

atgatcggag gtatttaggc agactgaa 208

Claims (9)

1. A primer composition for detecting polymorphism or genotype of SNP1477 in corn genome, wherein the primer composition consists of two single-stranded DNA shown as sequence 5 and sequence 6 in a sequence table; the SNP1477 is a SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table.

2. The primer composition according to claim 1, wherein: the molar ratio of the two single-stranded DNA is the same.

3. A product for detecting a polymorphism or genotype at SNP1477 site in the genome of maize, characterized in that: the product contains the primer composition of claim 1; the SNP1477 locus is one SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table.

4. A product according to claim 3, characterized in that: the product is a reagent or a kit.

5. A method for preparing the primer composition of claim 1, comprising the step of packaging the two single stranded DNAs of claim 1 separately.

6. A method of detecting a polymorphism or genotype at SNP1477 site in the genome of maize, comprising:

1) Performing PCR amplification by using the primer composition of claim 1 with genomic DNA of corn as a template to obtain a PCR amplification product;

2) Performing enzyme digestion on the PCR amplification product obtained in the step 1) by using restriction enzyme TaqI to obtain an enzyme digestion product, and determining polymorphism or genotype of the SNP1477 locus according to the size of the enzyme digestion product;

the SNP1477 locus is one SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table.

7. Use of a primer composition as claimed in claim 1 or 2 for any of the following:

a1 Use of the primer composition of claim 1 or 2 for detecting a polymorphism or genotype at SNP1477 site in corn genome;

a2 Use of the primer composition of claim 1 or 2 for detecting dwarf corn;

a3 Use of the primer composition of claim 1 or 2 in maize breeding and improvement;

the SNP1477 locus is one SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table.

8. Use of the product of claim 3 or 4 for any of the following:

b1 Use of the product of claim 3 or 4 for detecting a polymorphism or genotype at SNP1477 site in corn genome;

b2 Use of the product of claim 3 or 4 for detecting dwarf corn;

b3 Use of the product of claim 3 or 4 in maize breeding and improvement;

the SNP1477 locus is one SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table.

9. Use of the method of claim 6 for any of the following:

c1 Use of the method of claim 6 for detecting polymorphism or genotype at SNP1477 site in maize genome;

c2 Use of the method of claim 6 for detecting dwarf corn;

c3 Use of the method of claim 6 in maize breeding and improvement;

the SNP1477 locus is one SNP locus in a corn genome, the nucleotide type of the SNP locus is C or T, and the SNP locus is 1477 th nucleotide of a sequence 1 in a sequence table.