CN114774568A - 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. The technical scheme to be protected by the invention is a primer composition for detecting the polymorphism or genotype of SNP1477 in a corn genome, wherein the primer composition consists of two single-stranded DNAs shown as a sequence 2 and a sequence 3 in a sequence table; the SNP1477 is a SNP site in a maize genome, the nucleotide type of the SNP site is C or T, and the SNP site is the 1477 th nucleotide of a sequence 1 in a sequence table. The maize SNP1477 site dCAPS marker developed by the invention has an important effect on auxiliary site selection, and can be used for identifying whether maize 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 the breeding and improvement of the maize plant height.

Description

Molecular marker of maize half dwarf gene ZmD13 and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a molecular marker of a maize half-dwarf gene ZmD13 and application thereof.

Background

Corn is an important food crop and also an important animal feed and industrial raw material. In the past century, the increase in the yield per unit area of corn has not only relied on the increase in the yield per plant, but also on the increase in planting density, which is an important factor. The dense corn variety has the characteristics of proper plant height, compact plant type, developed root system, tough and tough stem, stress resistance, disease resistance and the like. The plant height is one of the key characters which influence the tightness of the corn variety. Reasonably reducing the plant height of the variety is beneficial to enhancing the lodging resistance of the corn, improving the planting density, improving the group permeability and optimizing the light energy utilization efficiency, thereby realizing the aim of improving the yield per unit area. Therefore, digging plant height regulating gene, breeding excellent short-stalk variety for popularization and application, and is an important direction for the breeding of corn with tolerance to breeding.

In the 60's of the last century, the discovery of rice and wheat dwarf genes rht1 and sd1 and the popularization and application of related dwarf derivative line varieties started a green revolution, and the yield of wheat and rice was greatly improved worldwide. In corn, the development of plant height regulating genes and the development of related functional markers have important significance for the breeding and application of short-stalk corn varieties.

The SNP marker is usually verified by Sanger (Sanger) sequencing method, but the cost is high and the flux is low. If the SNP is converted into the CAPS or dCAPs polymorphic codominant marker which can be verified by enzyme digestion, the SNP site detection can be efficiently and quickly realized through simple PCR amplification, restriction enzyme digestion and product gel electrophoresis. Due to the advantages, CAPS and dCAPs labeling technologies become important molecular labeling technologies in biological research and are widely applied to the fields of genetic map construction, germplasm identification, auxiliary breeding and the like.

Disclosure of Invention

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

In order to solve the above technical problems, the present invention first provides a primer composition for detecting a polymorphism or genotype of SNP1477 in the maize 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 site in a maize genome, the nucleotide type is C or T, and the SNP site is the 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 DNAs may be the same.

In order to solve the technical problems, the invention also provides a product for detecting the polymorphism or the genotype of the SNP1477 locus in the corn genome. The product contains the primer composition described above. The SNP1477 site is one SNP site in a corn genome, the nucleotide type is C or T, and the SNP site is the 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 of preparing the primer composition described above. The method may comprise the step of separately packaging the two single-stranded DNAs described above.

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

1) the genomic DNA of corn is used as a template, and the primer composition is used for PCR amplification to obtain a PCR amplification product.

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

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

The enzyme-digested product of the corn with the CC genotype at the site of the SNP1477 can be two bands (185bp and 23bp), 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 enzyme digestion product of the corn with the genotype of the SNP1477 site being CT can be three strips (185bp, 23bp and 208bp), and the nucleotide sequences of the three strips 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 enzyme-digested product of the corn with the genotype TT of the SNP1477 site can be a strip (208bp), and the nucleotide sequence of the strip can be respectively shown as a sequence 9 in a sequence table.

Any of the following uses of the primer compositions described above also fall within the scope of the present invention:

A1) use of the primer composition described above for detecting a polymorphism or genotype at a SNP1477 site in the maize genome.

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

A3) Use of a primer composition as described above in maize breeding and improvement.

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

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

B1) use of a product as described above for detecting a polymorphism or genotype at a SNP1477 locus in the maize genome;

B2) the application of the product in the detection of the dwarf corn is disclosed.

B3) Use of the product described above in maize breeding and improvement.

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

Any of the following uses of the above described method also fall within the scope of the invention:

C1) use of the method described above for detecting a polymorphism or genotype at a SNP1477 site in the maize genome.

C2) The method is applied to the detection of the dwarf corn.

C3) Use of the method described above in maize breeding and improvement.

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

The corn can be selected from hybrid progeny of a corn dwarf mutant d 13X corn inbred line B73, such as F2 or above; it can also be selected from the filial generation of maize dwarf mutant d 13X inbred line Chang 7-2, such as F2 generation or its progeny.

The invention discovers a recessive single-gene controlled semi-dwarf mutant dwerf 13(d13) in a maize EMS mutation mutant library, then clones a new gene ZmD13 participating in the regulation and control of the maize plant height, and discovers that the nucleotide type of the SNP1477 site of the gene ZmD13 is C or T and is a key site for generating a d13 dwarf phenotype; on the basis, the development of dCAPS markers at SNP1477 sites of the maize is carried out, the method has an important effect on auxiliary site selection, and can be used for identifying whether the maize 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 the breeding and improvement of the corn plant height.

Drawings

FIG. 1 shows phenotypic analysis of mutant d13 and Wild Type (WT). (A-D): mutant d13 compared to the WT phenotype; (E) the method comprises the following steps d13 vs WT internode length; (F) the method comprises the following steps d13 is compared to WT-type values, representing P < 0.01.

Fig. 2 is a CRISPR/Cas9 editing mutation of ZmD13 gene. (A) Editing the mutant versus Wild Type (WT) phenotype; (B) editing target sequencing analysis; (C) editing mutants compared to Wild Type (WT) plant height and ear height values, { character } represents P < 0.01; (D) editing mutants were compared to Wild Type (WT) internode length.

FIG. 3 is F₂And (4) a group construction process.

FIG. 4 shows a phenotype comparison of d13 with wild type. The height of the dwarf mutant d13 is about 50% of that of the wild type, each stem node is obviously shortened, and the length of the leaf is shorter than that of the wild type.

FIG. 5 shows dCAPS marker development.

FIG. 6 shows Wild Type (WT), d13 mutant and F₁The dCAPs primer of (1) is amplified and digested.

FIG. 7 is F₂Bands of restriction enzyme of the population.

FIG. 8 shows the restriction enzyme digestion bands of the mutant and breeding inbred lines.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Sources of corn material used in the examples of the invention:

f is obtained by hybridizing the mutant d13 with a maize B73 inbred line₂Segregating populations (fig. 3);

zheng 58, Chang 7-2, Mo17, B73, heddle 31, Qiqi 319, Huangzaoquan, Huang C, 178, Ye 478, Dan 340, Yu 330, Shen 137, PH4CV, PH6WC, CL11, NG5, HD568, 11DM124 and B195 of 20 breeding inbred lines of corn are stored in the laboratory, and related documents are as follows: li Yu, Wang Tianyu, formation of germplasm basis and backbone parents for corn breeding in our country, corn science 2010, 18 (5): 1-8; hu X, Wang H, et al.. transcriptotome profiling and composition of mail ear surgery degrading the spike and flow differentiation locations bmc genomics.2016nov 22; 17(1) 959.doi:10.1186/s 12864-016. 3296-8.PMID: 27875998.

Example I discovery and cloning of novel Gene ZmD13 for regulating and controlling the development of maize plant height

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

1.1 screening of maize plant height mutant d13

EMS mutagenesis is carried out on the maize inbred line Chang 7-2 material, and about 10,000 mutagenesis generations (M) are obtained after EMS mutagenesis ears are mature₁) Seed of M₁The seeds are sown in single seed, and the single spike is harvested after selfing. M₂20 seeds of the generation are planted into ears, and the character investigation is carried out from the seedling stage to the mature stage. The 5 semi-dwarf mutants were found in one of the ear rows and were designated dwarf13(d 13). Then selfing the d13 mutant ear to obtain M₃And M₄And the progeny phenotype observation shows that the semi-dwarf mutation character can be stably inherited.

The dwarf phenotype of the maize d13 mutant can be observed at the 5-leaf stage, and the jointing stage is more obvious. The height and ear position of the mature d13 plant are only about 50% of those of wild type Chang 7-2(WT in figure 1) (A in figure 1). Further investigation of the number and length of the nodes between mutant and wild type revealed that the number of d13 nodes was not significantly different from wild type WT, but the length of each node was shorter than that of the wild type (B and E in fig. 1). The shortening of the ear upper 3 and ear lower 4 is most significant. To M₄Other agronomic traits of wild-type WT and d13 mutants in the generation population were investigated and it was found that leaf blade and tassel major axis length were shortened in the d13 mutant in addition to dwarfing (C, D and F in FIG. 1).

1.2 genetic analysis and map cloning of 1.2ZmD13 Gene

Crossing d13 (male parent) with B73 (female parent) inbred line to construct F₂Segregating the population, and performing phenotype survey and chi-square test to show that the segregation ratio of the dwarf plants to normal plants is 3: 1 segregation law, the d13 mutant trait is controlled by a recessive monogene.

Extracting parent and F₂DNA of 78 d13 mutants in the population was isolated and the samples were genotyped using a targeted sequencing genotyping technique (20K GBTS). Through data analysis, the total number of available polymorphic SNP markers between parents is 7,029, the markers uniformly cover 10 chromosomes of corn, and the average number of the polymorphic SNP markers is about 3 SNPs per Mb. Calculating the SNP index (i.e. the genotype frequency of the mutant parent d13) of each SNP marker respectively, and analyzing the variation rule of the genome-wide SNP index. In the 1-18Mb interval of the short arm of chromosome 3, the marker SNP index value exceeds 0.5 and gradually approaches 1, which shows that the interval is linked with the target character and is a candidate segment. Analysis of the 78 samples for genotype changes in this region revealed that the minimal initial localization interval was between 4.1 and 6.0 Mb. Go to oneThe location population is expanded, 7 InDel markers with polymorphism between parents are developed, and the interval is narrowed to 200 kb. According to the prediction result of a Gramene (http:// Gramene. org /) website, 5 protein coding genes are also arranged in the interval. Sequencing analysis finds that a new gene for coding cytochrome P450 enzyme has a C-T mutation on the 8 th exon, and accords with the characteristic of EMS mutagenesis. The single base mutation forms a stop codon, the d13 mutant protein translation is terminated early, 10 amino acids are lacked compared with the wild type, the gene is presumed to be a candidate gene, and the candidate gene is named ZmD 13.

The CDS sequence of ZmD13 gene is shown in sequence 1 in sequence table, in sequence 1, y is c or t. 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 1.3 ZmD13 Gene

2 targets were selected on the active center heme binding domain (heme binding domain) sequence of ZmD13 protein using CRISPR/Cas9 basic vector (CPB vector) (Li C, et al., RNA-defined Cas9 as in vivo desired-target polypeptide in main, plant Biotechnol J.2017 Dec; 15(12):1566-1576.doi:10.1111/pbi.12739.Epub 2017May 12.PMID: 28379609), a grant-related document by researchers at the institute of crop science, academy of Chinese Agrology: Li. And constructing an expression cassette of double-target sgRNA and Cas9 protein, inserting the expression cassette into a binary expression vector CPB, and completing construction of a CRISPR/Cas9 knockout vector.

The two sgrnas targeted ZmD13 were sgRNA1 and sgRNA2, respectively. Wherein the nucleotide sequence of the sgRNA1 target is 5'-GAAGAGGCGGAAAACCGACG-3', and targets the 1044-1063 th position of the sequence 2 in the sequence table of the ZmD13 gene; the nucleotide sequence of the sgRNA2 target is 5'-GGCTTCACCCCTTTTGGCGG-3', and targets the 1324-th 1343-th position of the sequence 1 in the sequence table of the ZmD13 gene.

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

preparation of target fragment primers used:

TABLE 1 primer sequences

The target fragment U6-2 promoter is obtained by amplifying site1-U6-2-F (site2-U6-2-F) and site1-U6-2-R (site2-U6-2-R) primers. The target segment sgRNA fragment is obtained by amplifying a site1-sgRNA-F (site2-sgRNA-F) and a site1-sgRNA-R (site2-sgRNA-R) primer. The method comprises the following specific steps: performing PCR reaction by using a CPB carrier as a DNA template, wherein the amplification procedure is pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 20s, and extension at 68 ℃ for 20s, and 35 cycles; extending at 68 deg.C for 5min, and storing at 12 deg.C.

After detection by 1% agarose Gel electrophoresis, Gel recovery of the U6-2 promoter and sgRNA fragment was performed using the M5 Gel Extraction Kit.

The target fragment is prepared mainly by using an overlapping PCR technology, an overlapping fragment is obtained after overlapping PCR reaction, and the overlapping PCR product contains a target fragment, namely U6-2: gRNA (U6-2: gRNA1: sgRNA or U6-2: gRNA2: sgRNA) (the 300-802 th nucleotide of a sequence 3 in a sequence table or the 803-1305 th nucleotide of the sequence 3 in the sequence table).

Preparation and recovery of CPB enzyme digestion vector: the CPB vector was digested with HindIII in a total of 50. mu.L: 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 ddH₂O。

Connection and transformation of target fragment and enzyme digestion vector

Using a Hieff

The Plus One Step Cloning Kit (Shanghai assist in san Francisco Biotechnology Co., Ltd., Cat #10911es20) was ligated to the digested linear vector (i.e., the above-mentioned CPB digested vector) and the target fragment (i.e., the above-mentioned overlap PCR product) to obtain a recombinant vector, and after the recombinant vector was transformed into DH 5. alpha. competent cells, colony PCR was performed and sequencing was performed (primers: 5'-AACTGTAGAGTCCTGTTGTC-3' and 5' -CATTCGCC)ATTCAGGCTGC-3'), and the sequencing shows that the recombinant vector positive clone bacteria contain double target spots U6-2, gRNA1, sgRNA 6-2, gRNA2, sgRNA (sequence 3 in the sequence table) and an expression cassette of Cas9 protein (sequence 4 in the sequence table). Shake culturing positive monoclonal bacteria, extracting CRISPR-Cas9 recombinant plasmid ZmD13-gRNA1-gRNA2, transforming agrobacterium EHA105 strain to obtain recombinant agrobacterium EHA105/ZmD13-gRNA1-gRNA2, transforming corn B104 (Beijing Bomeixing Okorac Co., Ltd.) immature embryo with recombinant agrobacterium EHA105/ZmD13-gRNA1-gRNA2, and obtaining gene editing corn T₀Plant generation, T₀Inbreeding of corn generations to obtain T₁And (4) generation.

For gene editing T₀And T₁Carrying out PCR amplification detection and sequencing on target sequences of the generation plants by using primers 5'-GCGCCTAAACATCAGCAGA-3' and 5'-ACAACACCGAGGAGCACAC-3' to obtain 2 double homozygous mutants which are respectively named as ZmD13-cr1 and ZmD13-cr 2; the phenotype determination and observation are carried out on the 2 double homozygous mutants, and the results show that the phenotype of the 2 double homozygous mutants is highly similar to that of the d13 mutant, and the phenotype is represented by shortened stem nodes, dwarf plants, and the plant height and the ear position height are respectively about 61.9-69.5% and 90.3-92.9% of the wild type transgenic acceptor inbred line B104 (WT in figure 2) (table 2 and figure 2). According to the results, ZmD13 has the function of regulating the height of the corn plant.

TABLE 2 comparison of plant height and ear height of Gene editing mutants with wild-type B104

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

The nucleotide 1477 th site of the eighth exon of the maize cytochrome P450 enzyme gene ZmD13 gene, namely the sequence 1, is an SNP site, which is named as SNP1477, and the nucleotide at the SNP1477 site is C or T. The nucleotide at the SNP1477 site of the ZmD13 gene in the maize half-dwarf mutant dwarf13(d13) is T, and is a key site for generating the d13 dwarf phenotype. In the field identification of the plant height phenotype in the late pollen scattering stage of the corn plant, the plant height of the dwarf mutant d13 is about 50 percent of that of the 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 marker

dCAPS primer development and endonuclease selection are carried out on the SNP1477 site of the ZmD13 gene by using a dCAPS Finder 2.0Program (http:// helix. wustl. edu/dCAPS. html), a target fragment containing the SNP1477 mutation site is amplified, and the length of the amplified fragment is 208 bp. Corresponding downstream primers were designed using Primer Premier 5.0 software and Primer specificity was verified by Blast in NCBI. The primers were synthesized by Biotechnology engineering (Shanghai) GmbH, and the specific sequences are shown in Table 3:

TABLE 3 dCAPS primer sequences

dCAPS primer specificity and enzyme digestion fragment polymorphism detection

The nucleotide of SNP1477 locus in the homozygous wild type corn such as the corn B73 inbred line and the 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. Mutant base T is introduced at 2bp upstream of the SNP by using a dCAPS technology, so that 1 TaqI enzyme cutting site is introduced at the wild maize SNP1477 site (WT represents wild type in figure 5). Theoretically, a dCAPS primer amplification product of a wild-type SNP1477 site comprises 1 TaqI enzyme cutting 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 amplification product at SNP1477 site of mutant d13 has no enzyme cutting site, and the band length is 208bp (sequence 9 in the sequence table).

The dCAPS primer designed in the step 1 is used for pairing the short stalk mutant d13, the wild type inbred line Chang 7-2(WT) and the F thereof₁The hybrid was PCR amplified to obtain three PCR products, which were electrophoresed on 1% agarose gel to obtain single bands (208bp each), which were bright and clear, and correct in size (bands in lanes of PCR products in FIG. 6). The three PCR amplification products are cut by enzyme with TaqI restriction enzyme at 65 DEG CAfter 10 minutes of reaction, electrophoresis was carried out on a 3% agarose gel. The experimental results show that there are 3 types of bands A (208bp), B (185+23bp) and H (208+185+23bp) after the three PCR products are cut by enzyme, but the band of 23bp is basically invisible on the gel due to too short length. The d13 mutant band is A-type band (D13 in TaqI digested lane in FIG. 6), the Chang 7-2 band is B-type band (WT in TaqI digested lane in FIG. 6), F₁The hybrid bands are H-type bands (F in TaqI enzyme-digested lane in FIG. 6)₁). The dCAPS marker is proved to have strong specificity and co-dominant separation with characters, and can be used for detecting the mutation site in the d13 mutant, namely detecting the polymorphism of the SNP1477 site.

The experimental procedure was as follows:

extracting DNA of corn material:

plant DNA extraction kit (DP305) from Tiangen Biotechnology (Beijing) Ltd was used.

PCR amplification system and reaction conditions:

amplification system (enzyme for PCR amplification, Mf-002, Beijing Polymermai Biotech Co., Ltd.)

Template DNA* <1μg

2×M5 Taq HiFi PCR Mix 10μL

dCAPS1477-F(10μM) 0.5μL

dCAPS 477-R(10μM) 0.5μL

ddH₂O to make up to 20. mu.L

Reaction conditions are as follows:

and (3) an amplified product enzyme digestion system:

TaqI restriction enzyme (ThermoFisher Scientific, FD0674) recognition site 5'… T ^ CGA … 3';

enzyme digestion system:

PCR product < 1. mu.g

10x Buffer 2μL

TaqI 1μL

ddH2O to 20 μ L

Enzyme digestion program: storing at 65 deg.C for 10min and 4 deg.C.

Detecting the enzyme digestion fragments by gel electrophoresis:

3% agarose gel, voltage 160V, electrophoresis for 40 minutes.

dCAPS primer labeling at F₂Detection in a segregating population

24 parts of F obtained by hybridizing the mutant d13 with maize B73 inbred line by using dCAPS primer marker at SNP1477 site₂The segregating population (figure 3) performs genotype detection of the SNP1477 site, the detection method is the same as step 2. From the results of FIG. 7, it can be seen that at F₂Of the 17 plants of the population, which were similar to the normal maize B73 wild-type plant, 6 detected bands were B (185+23bp) (F in FIG. 7)₂-3、F₂-6、F₂-10、F₂-12、F₂-21、F₂-24, indicating the 6 parts of F₂The material is homozygous wild type (AA) with CC genotype at SNP1477 locus, and the detection band type of 11 materials is H (208+185+23bp) (F in figure 7)₂-1、F₂-4、F₂-5、F₂-7、F₂-8、F₂-9、F₂-15、F₂-17、F₂-18、F₂-20、F₂-22), indicating that 11 parts of F₂The material is heterozygous (Aa) with the genotype of SNP1477 locus being CT; in addition, 7 short-stalk mutant materials were all of type A (208bp) after digestion (F in FIG. 7)₂-2、F₂-11、F₂-13、F₂-14、F₂-16、F₂-19、F₂-23), indicating that 7 parts of F₂The material is homozygous mutant (aa) with genotype at SNP1477 site being TT. For 24 parts of F₂The plant heights were measured and calculated (table 4) and the average plant height of homozygous wild type (CC) was found to be: 253.5; the average plant height of the heterozygous wild (CT) is 258.0; the average plant height of homozygous mutant (TT) is 166.6 cm, only 65.7% and 64.6% of homozygous and heterozygous wild type, and the difference is very significant (t-test, p)<0.01). Therefore, the genotype detection result of the dCAPS primer molecular marker and the accuracy rate of the phenotype reach 100.0%. F₂Genotype distribution in segregating populations meets 1:2:1(χ)²<3.84), which complies with Mendelian inheritance rule, againThe maize dwarf trait is verified to be controlled by a recessive single gene.

TABLE 4.F₂Plant height and genotype of group plants

Detection of dCAPS primer marker in maize backbone inbred line

And further detecting the backbone inbred lines in 20 corn breeding lines by using dCAPS primers at SNP1477 sites, wherein the detection method is the same as the step 2. The detection result shows that the banding patterns of the products after enzyme digestion are all A types, namely the genotypes of the SNP1477 loci of the 20 parts of maize inbred lines are all homozygous wild types (AA), and the backbone inbred lines do not contain mutant SNP loci (fig. 8). Therefore, the d13 dwarf mutant can be used for carrying out high genetic improvement on maize plants to create dwarf germplasm, and the development of dCAPS marker at SNP1477 locus plays an important role in auxiliary locus 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 the breeding and improvement of the maize plant height.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within 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 reference to specific embodiments, it will be appreciated that the invention can 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 use of some of the essential features is made possible within the scope of the claims attached below.

Sequence listing

<110> institute of crop science of Chinese academy of agricultural sciences

Molecular marker of <120> maize half 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. The primer composition is used for detecting the polymorphism or genotype of SNP1477 in a corn genome, and consists of two single-stranded DNAs shown as a sequence 5 and a sequence 6 in a sequence table; the SNP1477 is a SNP site in a maize genome, the nucleotide type is C or T, and the SNP site is the 1477 th nucleotide of a sequence 1 in a sequence table.

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

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

4. The product of claim 3, wherein: the product is a reagent or a kit.

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

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

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

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

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

7. Use of the primer composition of claim 1 or 2, wherein the primer composition is selected from the group consisting of:

A1) use of the primer composition as set forth in claim 1 or 2 for detecting a polymorphism or genotype at a SNP1477 site in the maize genome;

A2) the 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 for corn breeding and improvement;

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

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

B1) use of the product of claim 3 or 4 for detecting polymorphisms or genotypes at the SNP1477 locus in the maize genome;

B2) the use of the product of claim 3 or 4 for the detection of dwarf corn;

B3) use of the product of claim 3 or 4 in corn breeding and improvement;

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

9. The method of claim 6, wherein any one of the following uses:

C1) use of the method of claim 6 for detecting a polymorphism or genotype at a SNP1477 site in the maize genome;

C2) the method of claim 6, applied to the detection of dwarf corn;

C3) use of the method of claim 6 in maize breeding and improvement;

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

Images