Kasp marker PH-03-KASP-106 closely linked with corn plant height and application of Kasp marker PH-03-KASP-106

Abstract

The invention relates to the technical field of plant molecular breeding, in particular to a Kasp marker PH-03-KASP-106 closely linked with corn plant height and application of the Kasp marker PH-03-KASP-106. The marker comprises a single nucleotide polymorphism (SNP) site located at the 163964821 position of a corn No.3 chromosome Chr3 and a KASP molecular marker primer group, a corn genome DNA is subjected to PCR amplification through the specific primer group, and a Kasp marker PH-03-KASP-106 molecular marker is obtained. TT dwarf stem and CC high stem genotypes are distinguished according to the difference of fluorescence signals. The method can be used for early identification and screening of corn plant height characters and molecular marker-assisted breeding, can improve the breeding efficiency of dwarf corn germplasm, enhance the selection accuracy and determine the breeding direction, is simple and convenient to operate, has high cost effectiveness and is suitable for breeding research and production practice of corns with different scales.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular breeding, in particular to a Kasp marker PH-03-KASP-106 tightly linked to corn plant height and an application thereof. Background Art

[0002] In the field of maize molecular breeding, plant height, a key agronomic trait affecting yield, lodging resistance, and planting density, has long been a core goal of breeding research. Traditional breeding methods rely on phenotypic selection, which has limitations such as long breeding cycles, significant environmental interference, and insufficient genetic information. In recent years, the development of marker-assisted selection (MAS) technology has provided an important tool for the early identification and targeted improvement of maize plant height traits. However, existing technologies still have the following shortcomings: 1. InDel and SSR molecular marker technology, represented by Reference 1 (CN109337998B, which discloses methods for developing and applying InDel6 and SSR229 molecular markers tightly linked to maize plant height), has achieved preliminary localization of plant height-related genes through BSA (Block Segregant Analysis) and genome resequencing. However, its technical approach has significant drawbacks: (1) Low detection efficiency: Polymorphism analysis relies on gel electrophoresis, which is cumbersome and time-consuming. In addition, the electrophoresis bands are easily affected by experimental conditions, resulting in insufficient stability of the results. (2) Insufficient marker density: The distribution density of InDel and SSR markers in the genome is limited, making it difficult to achieve high-precision linkage analysis, especially in the target gene region where markers may be missing or linkage may be loose; (3) High breeding application costs: Multiple rounds of population screening and exchange of single plant identification are required to verify the effectiveness of the marker, which significantly increases the breeding cycle and labor costs.

[0003] 2. Comparative Document 2 (CN119859638A discloses a corn plant type regulatory gene ZmEXO1 and its application) reveals the regulatory effect of the ZmEXO1 gene on plant type through gene editing technology, but its technical approach has the following problems: (1) High technical threshold: It relies on gene editing tools such as CRISPR / Cas9, which is complex to operate and requires the construction of a transgenic system, facing regulatory restrictions and public acceptance challenges; (2) Functional redundancy risk: Gene editing may lead to non-target effects. For example, mutations in ZmEXO1 may trigger linkage variations in other agronomic traits, requiring additional verification of phenotypic stability. (3) Limited scope of application: It is mainly used to verify the function of a single gene and is difficult to be directly applied to the improvement of complex plant height traits regulated by multiple genes.

[0004] 3. Although KASP (competitive allele-specific PCR) technology has been gradually applied to molecular breeding, existing markers (such as some SNP markers) still have the following defects: (1) Low efficiency of marker development: Candidate sites need to be screened through genome-wide association analysis (GWAS) or QTL mapping, which has a long development cycle and relies on large-scale population data; (2) Unclear phenotypic association: The genetic correlation between some SNP markers and plant height has not been verified, resulting in insufficient accuracy of marker-assisted selection; (3) The detection system has poor universality: primer design and reaction conditions lack standardization, and the comparability of results between different laboratories is low. Summary of the Invention

[0005] The object of the present invention is to provide a Kasp marker PH-03-KASP-106 tightly linked to maize plant height and its application, so as to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides a Kasp marker PH-03-KASP-106 tightly linked to maize plant height, comprising a single nucleotide polymorphism (SNP) site tightly linked to maize plant height trait and a KASP molecular marker primer set; The single nucleotide polymorphism (SNP) site is located at position 163964821 of chromosome 3 of maize, Chr3, and its polymorphism is expressed as base T / C, wherein the C allele is associated with the dwarf corn trait, and the T allele is associated with the tall corn trait.

[0007] Preferably, the KASP molecular marker primer set is used to detect the single nucleotide polymorphism (SNP) site, and the KASP molecular marker primer set includes: Forward primer 1: SEQ ID NO: 1 (PH-03-KASP-106F1) Forward primer 2: SEQ ID NO: 2 (PH-03-KASP-106F2) Reverse primer: SEQ ID NO: 3 (PH-03-KASP-106R).

[0008] Preferably, the amplification region of the KASP molecular marker primer set covers the Chr3:163964621-163965021 interval, and distinguishes the TT short-stem and CC tall-stem genotypes by differences in fluorescence signals.

[0009] Preferably, the reagent for detecting corn plant height comprises a KASP molecular marker primer set, a 2×Probe Mix A solution, and ddH2O, wherein: The concentrations of forward primer 1 and forward primer 2 were independently 4–10 μmol / L; The concentration of the reverse primer was 4-10 μmol / L.

[0010] Preferably, detecting the height of corn plants specifically includes the following steps: The KASP molecular marker primer set was used to perform PCR amplification on maize genomic DNA, and the fluorescence signal was detected by KASP genotyping to determine the plant height trait according to the following rules: TT genotype: orange-red fluorescent signal, corresponding to the dwarf trait; CC genotype: blue fluorescent signal, corresponding to the tall stem trait; CT genotype: heterozygous signal.

[0011] Preferably, the reaction system for PCR amplification is 10 μL, which includes: 1-3 μL of maize genomic DNA, concentration 50-100 ng / μL; KASP molecular marker primer set 0.1-0.15 μL; 2×ProbeMixA solution 4-6 μL; Add ddH2O to make up to 10 μL.

[0012] Preferably, the addition ratio of forward primer 1:forward primer 2:reverse primer in the KASP molecular marker primer set is 2:2:5.

[0013] Preferably, the steps of PCR amplification are: Pre-denaturation at 95°C for 10 minutes; Denaturation at 95°C for 20 seconds and annealing at 61°C for 40 seconds were performed for 10 cycles; Denaturation at 95°C for 20 seconds and annealing at 55°C for 40 seconds were performed for 31 cycles; Keep at 25℃ for 10 minutes.

[0014] On the other hand, the present invention also provides an application of a Kasp marker PH-03-KASP-106 in corn breeding, which is used for early identification, screening or molecular marker-assisted breeding of corn plant height traits to improve the breeding efficiency of dwarf corn germplasm and for screening / preparing dwarf corn inbred lines.

[0015] Preferably, the method for preparing dwarf corn inbred lines is to use single nucleotide polymorphism (SNP) sites or the KASP marker PH-03-KASP-106 to selectively breed corn inbred lines with TT genotype.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) Improve breeding efficiency: With the help of Kasp marker PH-03-KASP-106, in the early stage of corn breeding, based on the detection of specific SNP sites and primer sets, the plant height trait of corn can be accurately identified, and dwarf corn germplasm can be quickly screened out, which greatly shortens the breeding cycle, improves the efficiency of dwarf corn germplasm selection, and accelerates the genetic selection and improvement process of corn varieties.

[0017] (2) Enhanced selection accuracy: This marker can accurately distinguish TT dwarf, CC tall, and CT heterozygous genotypes based on the difference in fluorescence signals. Breeders can accurately select plants of the target genotype based on the test results, effectively avoiding the errors of traditional breeding that rely on phenotypic selection, enhancing selection accuracy, and improving breeding success rates.

[0018] (3) Clarify the direction of molecular marker-assisted breeding: provide a basis for the utilization of excellent allele variation related to corn plant height. In molecular marker-assisted breeding, help breeders to perform more targeted hybridization, backcrossing and other operations based on molecular marker information, and realize the directional breeding of corn plant height traits. For example, the directional breeding of TT genotype corn inbred lines to prepare dwarf corn inbred lines will improve the scientific nature and operability of breeding work.

[0019] (4) Easy to operate and cost-effective: The detection method is relatively simple to operate, requiring only DNA extraction, PCR amplification, and genotyping. Furthermore, the parameters such as primer set concentration, reaction system, and amplification steps are clear, making it easy to standardize and scale up. While ensuring detection accuracy, it can reduce detection costs and improve detection efficiency, making it suitable for corn breeding research and production practices of different scales. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they provide further detailed explanation, but do not constitute a limitation of the present invention.

[0021] Figure 1 is the normal distribution diagram of plant height traits; Figure 2 Chromosome distribution map of SNP markers; Figure 3 QQ plot and Manhattan plot for genome-wide association analysis of maize plant height; Figure 4 To analyze the allelic variation effects of two important SNP sites related to plant height; Figure 5 Comparison of some results of Sanger sequencing of 300 maize candidate genes; Figure 6 The results of KASP molecular marker typing for plant height of 256 maize samples; Figure 7This is the effect value analysis of plant height of KASP haplotype; Figure 8 Table 1 is the result of scanning 256 maize plant height KASP molecular markers; Figure 9 Table 1 is the result of scanning 256 maize plant height KASP molecular markers; Figure 10 This is the KASP genotyping and phenotyping statistical results table. DETAILED DESCRIPTION

[0022] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0023] The SNP provided by the present invention is located at position 163964821 on maize chromosome 3 and has a polymorphism of T / C. When the base is C, the maize plant height trait is short; when the base is T, the maize plant height trait is tall. This SNP can be used to distinguish tall from short maize varieties.

[0024] The present invention uses a KASP molecular marker primer set to select for the plant height trait of corn. Sample identification can be completed through DNA extraction, PCR-specific amplification, and KASP genotyping detection, and dwarf corn can be obtained. The molecular marker is effective in identifying corn plant height. The molecular marker-assisted selection of the present invention can improve the efficiency of dwarf corn germplasm breeding, provide a basis for the utilization of excellent allelic variations related to corn plant height, and accelerate the breeding process.

[0025] The present invention provides 1,149 phenotypically diverse and representative DH lines selected from 5,572 maize DH lines. These lines were planted in an experimental field in Guangxing Village, Yangshu Street, Acheng District, Harbin City, Heilongjiang Province (126°53′8.73″E, 45°29′39.88″N) in 2023 and 2024, respectively. Each material was planted in two rows with a row length of 2.5 m. Twenty-one major agronomic traits and yield traits were investigated according to standards, and data on traits such as plant height were recorded and organized. After simplified genome resequencing of each material, the reference genome was aligned and spliced to obtain SNP polymorphism markers. Based on GWAS association analysis of the plant height trait data, the associated major effect SNPs were obtained, and haplotype analysis was performed on the major effect SNPs. Based on field phenotypes, 150 varieties of tall and dwarf maize inbred lines were selected. The major SNP polymorphism was verified by Sanger sequencing, and the KASP marker in the sequence was obtained. KASP primers were designed based on the SNP, and gel-free fluorescent polymerase chain reaction was used to distinguish the plant height haplotypes of the maize inbred lines.

[0026] The present invention relates to specific primers that can effectively genotype maize plant height and detect differences in SNP polymorphisms. The maize plant height trait KSAP marker provided by the present invention can be used in molecular marker-assisted breeding for maize plant height traits, and has important theoretical and practical guiding significance for accelerating the genetic selection and improvement of maize varieties.

[0027] The goal of this study is to locate SNPs associated with maize plant height and, based on the sequence information from these sites, develop a KASP-specific marker for identifying maize plant height. This molecular marker can be used to predict maize plant height, providing molecularly assisted technology support for early identification and screening of maize plant height traits. Example 1: Investigation of plant height traits and phenotypic data analysis of maize inbred lines

[0028] 1,149 high-quality maize inbred lines bred over the past 15 years, provided by the Shenzhen Genomics Center of the Chinese Academy of Agricultural Sciences, were grown under field conditions in Acheng, Harbin, Heilongjiang Province, in 2023 and 2024. A randomized block design was used, with two rows of each variety planted, each 3 m long, with a row spacing of 0.65 m and a plant spacing of 0.2 m. Fertilization and watering were the same as for conventional field management. Plant height was measured at maturity, excluding the first plant in each row. Five representative plants of each variety were selected to measure the distance from the ground to the top of the corn. Data on traits such as plant height were recorded and organized. Phenotypic data from the two environments were statistically analyzed using Microsoft Excel 2022 and IBM SPSS Statistics V27.0. Normal distribution was assessed based on the coefficient of variation, skewness, and kurtosis. Finally, frequency distribution histograms were plotted using Origin 2021 software to test the normal distribution of the phenotypic data. Statistical analysis of maize plant height showed that the mean values under the two environments ranged from 216.66 to 218.46 cm, the phenotypic variation ranged from 127.00 to 317.00 cm, and the coefficient of variation ranged from 11.95% to 14.14%. The coefficient of variation in each environment exceeded 10%, indicating that the maize inbred line population had a relatively rich phenotypic variation in plant height. The absolute values of the skewness and kurtosis of the plant height trait were both less than 1, and the data distribution curve conformed to a normal distribution, indicating that the plant height data conformed to the quantitative trait characteristics (such as Figure 1 ), Figure 1 a, b, and c represent Figure 1 Plant height distribution diagrams of 2023, 2024 and BLUP. PH-2023H, PH-2024H and PH-BLUP represent Figure 1 Phenotypic data for Harbin plant height in 2023 and 2024, and field phenotypic data for Harbin plant height using BLUP. Phenotypic data for maize plant height were analyzed using the 1me4 R package, along with the variance of each influencing factor to estimate broad-sense heritability. The heritability of plant height was 86%, indicating that it is primarily influenced by genetic factors. Example 2: Extraction of maize genomic DNA and library construction and sequencing

[0029] For the maize inbred line materials in Example 1, the specific library construction method is as follows: (1) Weigh 1.0 g of fresh leaves, chop them into small pieces, place them in a mortar, grind them with liquid nitrogen, add 3 mL of 1.5× CTAB, grind them into a homogenous slurry, transfer them to a 15 mL centrifuge tube, then add 1 mL of 1.5× CTAB to the mortar to rinse, and transfer them to the centrifuge tube. After mixing, place them in a 65°C water bath for 30 min, shaking them gently from time to time.

[0030] The formula of 1.5×CTAB is as follows (1L):

[0031] Add deionized water to make up to 1 L, and add mercaptoethanol to a final concentration of 0.2% (2 ml) before use.

[0032] (2) After cooling to room temperature, add an equal volume of chloroform / isoamyl alcohol (24:1) and mix gently until the lower layer turns dark green.

[0033] (3) Centrifuge at 4200 rpm for 10 minutes. Transfer the upper aqueous phase to a new 15 mL centrifuge tube, add 2 volumes of pre-cooled anhydrous ethanol, mix and let stand for 5 minutes. Place at -20°C for 30 minutes to precipitate DNA.

[0034] (4) Centrifuge at 4200 rpm for 10 min, discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate once, invert the centrifuge tube to dry the DNA, and add 50 μL of TE to dissolve the DNA.

[0035] (5) Detect the DNA concentration and adjust it to 20 ng / ul with water.

[0036] (6) FBI-seq method for library construction (Zhao et al., 2023) Example 3: GWAS analysis of maize plant height to obtain significant SNPs and candidate genes

[0037] All off-line sequencing data were processed and analyzed using high-performance computer servers. Raw data processing: Raw PE (pair-end) sequencing data were quality-assessed using FastQC and then quality-controlled using BWA. Sequencing reads were aligned to the reference genome (B73v4), and SNP detection was performed using GATK. After sample- and variant-level quality control filtering, 57,849 high-quality SNP markers (minor allele frequency > 0.05, missing data < 20%) were identified, ensuring the accuracy and reliability of the analysis results. To better understand population structure and genetic background, phylogenetic trees were constructed using iqTree software. Principal component analysis (PCA) of genome-wide SNP data was performed using Plink software, and population structure analysis was performed using Faststructure software to clarify the genetic structure within the population. Genome-wide association analysis of plant height and its BLUP values was performed using the previously selected high-quality SNPs. Association analyses between SNP markers and various traits were performed using mixed linear models in GEMMA, combining genotype, phenotype, population structure, and kinship matrix. All SNPs with p < 1.7286e-5 were extracted from the GWAS result file using awk and converted to a BED format file (Chr, Start, End). Two major SNPs related to plant height were obtained based on the two-year GWAS data. The bedtoolsintersect tool was used to compare the significant SNPs and their upstream and downstream 100 kb regions with the B73 RefGen_v4 GFF gene annotation file to screen candidate genes. The results are shown in Figure 2-Figure 4 , Figure 3 .a and b are the QQ plot and Manhattan plot of plant height in 2023; Figure 3 c and d are the QQ plot and Manhattan plot of plant height in 2024; Figure 3 e and f are the QQ plots and Manhattan plots of plant height under the BLUP environment. 2023PH, 2024PH, and BLUP PH represent the plant height in 2023, 2024, and BLUP environment, respectively. Example 4: Association analysis of candidate genes for maize plant height and mining of new SNP sites and KASP markers

[0038] Based on field phenotypes, 150 tall and dwarf maize inbred lines, as well as maize cultivar B73, were selected. Genomic DNA from leaves was extracted using the CTAB method. Full-length sequences of candidate genes (including the 5' UTR, 3' UTR, and exon sequences) were amplified and subsequently Sanger sequenced for haplotype analysis. DNA sequences were amplified using a segmented amplification method. Primers were designed using the free online primer design tool NCBI and synthesized by Shenzhen Sangon Biotechnology Co., Ltd. The amplification and sequencing primers are as follows:

[0039] The sequences obtained by sequencing were submitted to Snapgene software for multiple sequence alignment. SNP sites with missing values greater than 20% and minor allele frequency (MAF) ≤ 5% were removed to verify the authenticity of the SNP variant site. At the same time, a new SNP marker was discovered, located at 163964821 on chromosome 3 of maize. Single gene association analysis was then performed using Tassel software. Haploview software was used to output the haplotype typing results in nex format containing only polymorphic sites (including indels) and to draw an LDblock diagram. Excel software was used to calculate the distribution of each haplotype in different subpopulations and Origin software was used for data visualization. The results are shown in the figure. Figure 4 and Figure 5 , Figure 4 a and Figure 4 b is the allelic variation effect analysis of two important SNP sites. 2023, 2024, and BLUP represent the plant height in 2023, 2024, and BLUP environment, respectively. Figure 5 The Sanger sequencing results of 300 maize candidate genes showed that Chr3:163964821 in dwarf maize was T and Chr3:163964821 in tall maize was C. Example 5: Development and validation of KASP markers for maize plant height

[0040] Based on the results of candidate gene association analysis, we screened for SNP variants that could differentiate plant height traits among different maize inbred lines. We then developed KASP molecular markers that could rapidly identify the two haplotypes at these sites.

[0041] The details are as follows: (1) DNA was extracted from 249 maize inbred lines sequenced by Sanger sequencing using the CTAB method; (2) Using DNA as a template, KASP primers were used for fluorescence polymerase chain reaction genotyping. KASP primers were designed based on the SNP at 163964821 on chromosome 3 of maize. The primers are as follows:

[0042] The PCR amplification reaction system (10 μl) consists of: 2 μl of 4-50 ng / μl genomic DNA, 0.14 μl of primer mix (prepared by mixing 6 μl forward primer 1 + 6 μl forward primer 2 + 15 μl reverse primer + 23 μl ddH2O), 5 μl of 2x ProbeMix A solution, and 3 μl of ddH2O. The amplification program of the PCR amplification reaction was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing at 61°C for 40 s, 10 cycles; denaturation at 95°C for 20 s, annealing at 55°C for 40 s, 31 cycles; holding at 25°C for 10 min; and storage at 4°C.

[0043] After amplification, KASP detection was performed based on the operating instructions of the AQP genotyping system. The PCR program on the ABI7500 qPCR instrument was set to 35°C for 30 seconds. The result file was exported, and the genotype was further determined based on the sample cluster. The result analysis was performed on the Taqman Genotyper Software software. The fluorescence values corresponding to HEX and FAM of each PCR reaction well were obtained and divided by the value of the reference dye (ROX) of the well. The data fluorescence values were standardized to obtain the relative fluorescence values corresponding to HEX and FAM of each PCR reaction well (FAM fluorescent label sequence was observed and read at an excitation light wavelength of 485nm and an emission light wavelength of 520nm, and the HEX fluorescent label sequence was observed and read at an excitation light wavelength of 528nm and an emission light wavelength of 560nm). The samples were clustered according to the relative fluorescence values. The test results are as follows: Figure 6 shown.

[0044] Depend on Figure 6 It can be seen that there are two genotypes at this site. KASP primers were designed based on the SNP at Chr3:163964821 in maize. 256 samples were selected from 300 maize candidate gene Sanger sequencing samples for KASP molecular marker typing. The results showed that the KASP marker can distinguish the two genotypes at Chr3:163964821.

[0045] Figure 6 The orange-red color is TT, the blue color is CC, and the gray color is NTC control.

[0046] The correspondence between the plant height traits of the corn materials and the typing results and the degree of agreement between the plant height traits of the corn materials and the typing results were analyzed in Example 4. Figure 8 、 Figure 10 and Figure 2 shown.

[0047] according to Figure 8 、 Figure 10and Figure 6 、 Figure 7 As can be seen, when the fluorescent signal of the amplified product is orange-red, the maize plant height trait is identified as dwarf, corresponding to the genotype TT; when the fluorescent signal of the amplified product is blue, the maize plant height trait is identified as homozygous for tall plant height, i.e., CC. The KASP experimental results of Example 5 are consistent with the actual plant height traits of the tested samples. 145 T / T materials (orange dots) and 111 C / C materials (blue dots) were detected. The tall plant height trait was 100% consistent with the actual plant height traits of the tested materials, and the dwarf plant height trait was 100% consistent with the actual plant height traits of the tested materials. The KASP experimental results and sequencing results were also consistent. Overall, among the 256 tested samples, the KASP test results were 100% consistent with the traits, and the KASP test results were 100% consistent with the sequencing results. This shows that using this molecular marker to perform KASP experiments on the tested materials can effectively detect their genotypes, thereby completing the identification of germplasm.

[0048] The present invention uses a specific Kasp marker, PH-03-KASP-106, for corn plant height detection, which has significant beneficial effects in corn breeding and other aspects, as follows: (1) Improve breeding efficiency: With the help of Kasp marker PH-03-KASP-106, in the early stage of corn breeding, based on the detection of specific SNP sites and primer sets, the plant height trait of corn can be accurately identified, and dwarf corn germplasm can be quickly screened out, which greatly shortens the breeding cycle, improves the efficiency of dwarf corn germplasm selection, and accelerates the genetic selection and improvement process of corn varieties.

[0049] (2) Enhanced selection accuracy: This marker can accurately distinguish TT dwarf, CC tall, and CT heterozygous genotypes based on the difference in fluorescence signals. Breeders can accurately select plants of the target genotype based on the test results, effectively avoiding the errors of traditional breeding that rely on phenotypic selection, enhancing selection accuracy, and improving breeding success rates.

[0050] (3) Clarify the direction of molecular marker-assisted breeding: provide a basis for the utilization of excellent allele variation related to corn plant height. In molecular marker-assisted breeding, help breeders to perform more targeted hybridization, backcrossing and other operations based on molecular marker information, and realize the directional breeding of corn plant height traits. For example, the directional breeding of TT genotype corn inbred lines to prepare dwarf corn inbred lines will improve the scientific nature and operability of breeding work.

[0051] (4) Easy to operate and cost-effective: The detection method is relatively simple to operate, requiring only DNA extraction, PCR amplification, and genotyping. Furthermore, the parameters such as primer set concentration, reaction system, and amplification steps are clear, making it easy to standardize and scale up. While ensuring detection accuracy, it can reduce detection costs and improve detection efficiency, making it suitable for corn breeding research and production practices of different scales.

[0052] The basic principles, main features, and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited to the above-described embodiments. The above-described embodiments and descriptions are merely preferred examples of the present invention and are not intended to limit the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention claimed.

Claims

1. A Kasp marker PH-03-KASP-106 tightly linked to maize plant height, characterized in that: Including single nucleotide polymorphism (SNP) sites closely linked to maize plant height traits and KASP molecular marker primer sets; The single nucleotide polymorphism (SNP) site is located at position 163964821 of chromosome 3 of maize, Chr3, and its polymorphism is expressed as base T / C, wherein the C allele is associated with the dwarf corn trait, and the T allele is associated with the tall corn trait.

2. The Kasp marker PH-03-KASP-106 tightly linked to maize plant height according to claim 1, characterized in that The KASP molecular marker primer set is used to detect the single nucleotide polymorphism (SNP) site, and the KASP molecular marker primer set includes: Forward primer 1: SEQ ID NO: 1 (PH-03-KASP-106F1) Forward primer 2: SEQ ID NO: 2 (PH-03-KASP-106F2) Reverse primer: SEQ ID NO: 3 (PH-03-KASP-106R).

3. The Kasp marker PH-03-KASP-106 tightly linked to maize plant height according to claim 1, characterized in that The amplification region of the KASP molecular marker primer set covers the Chr3:163964621-163965021 interval, and distinguishes the TT short-stem and CC tall-stem genotypes by fluorescence signal differences.

4. The Kasp marker PH-03-KASP-106 tightly linked to maize plant height according to claim 1, characterized in that The reagents for detecting corn plant height include a KASP molecular marker primer set, a 2×Probe Mix A solution, and ddH2O. The KASP molecular marker primer set includes: The concentrations of forward primer 1 and forward primer 2 were independently 4–10 μmol / L; The concentration of the reverse primer was 4-10 μmol / L.

5. The Kasp marker PH-03-KASP-106 tightly linked to maize plant height according to claim 1, characterized in that The specific steps for detecting corn plant height include the following: The KASP molecular marker primer set was used to perform PCR amplification on maize genomic DNA, and the fluorescence signal was detected by KASP genotyping to determine the plant height trait according to the following rules: TT genotype: orange-red fluorescent signal, corresponding to the dwarf trait; CC genotype: blue fluorescent signal, corresponding to the tall stem trait; CT genotype: heterozygous signal.

6. The Kasp marker PH-03-KASP-106 tightly linked to maize plant height according to claim 1, characterized in that The PCR amplification reaction system is 10 μL, which includes: 1-3 μL of maize genomic DNA, concentration 50-100 ng / μL; KASP molecular marker primer set 0.1-0.15 μL; 2×ProbeMixA solution 4-6 μL; Add ddH2O to make up to 10 μL.

7. The Kasp marker PH-03-KASP-106 tightly linked to maize plant height according to claim 1, characterized in that The addition ratio of forward primer 1:forward primer 2:reverse primer in the KASP molecular marker primer set is 2:2:

5.

8. The Kasp marker PH-03-KASP-106 tightly linked to maize plant height according to claim 1, characterized in that The steps of the PCR amplification are: Pre-denaturation at 95°C for 10 minutes; Denaturation at 95°C for 20 seconds and annealing at 61°C for 40 seconds were performed for 10 cycles; Denaturation at 95°C for 20 seconds and annealing at 55°C for 40 seconds were performed for 31 cycles; Keep at 25℃ for 10 minutes.

9. Use of the Kasp marker PH-03-KASP-106 according to any one of claims 1 to 8 in corn breeding, for early identification and screening of corn plant height traits or molecular marker-assisted breeding, to improve the breeding efficiency of dwarf corn germplasm, and for screening / preparing dwarf corn inbred lines.

10. The use of the Kasp marker PH-03-KASP-106 tightly linked to maize plant height according to claim 9, characterized in that: The method for preparing the dwarf corn inbred line is to utilize the single nucleotide polymorphism (SNP) site or the KASP marker PH-03-KASP-106 to directionally select the corn inbred line with the TT genotype.

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