Kasp marker ph-03-kasp-106 closely linked to corn plant height and application thereof

Abstract

The present application relates to the technical field of plant molecular breeding, and particularly relates to a Kasp marker PH-03-KASP-106 closely linked to corn plant height and application thereof, the marker comprises a single nucleotide polymorphism SNP site located at the position 163964821 of the 3rd chromosome of corn Chr3 and a KASP molecular marker primer set, PCR amplification of corn genomic DNA is carried out through the specific primer set, and the TT dwarf, CC high rod genotypes are distinguished according to fluorescence signal differences. The present application can be used for early identification, screening and molecular marker assisted breeding of corn plant height traits, can improve the efficiency of dwarf corn germplasm breeding, enhance the selection accuracy, clarify the breeding direction, and is simple in operation, high in cost-effectiveness, and suitable for corn breeding research and production practice of different scales.

Description

Technical Field

[0001] This invention relates to the field of plant molecular breeding technology, and more specifically, to a Kasp marker PH-03-KASP-106 closely linked to maize plant height and its application. Background Technology

[0002] In the field of maize molecular breeding, plant height, as a key agronomic trait affecting yield, lodging resistance, and planting density, has always been a core objective of breeding research for its precise improvement. Traditional breeding methods rely on phenotypic selection, which has limitations such as long cycles, significant environmental interference, and insufficient analysis of genetic information. In recent years, the development of molecular marker-assisted selection (MAS) technology has provided an important tool for the early identification and targeted improvement of maize plant height, but existing technologies still have the following shortcomings:

[0003] I. While InDel and SSR molecular marker technologies, exemplified by comparative document 1 (CN109337998B, which discloses the development method and application of InDel6 and SSR229 molecular markers closely linked to maize plant height), have achieved preliminary localization of plant height-related genes through BSA (group segregation analysis) and genome resequencing, their technical approach has significant shortcomings:

[0004] (1) Low detection efficiency: It relies on gel electrophoresis for polymorphism analysis, which is complicated and time-consuming. Furthermore, the electrophoretic bands are easily affected by experimental conditions, resulting in insufficient stability of the results.

[0005] (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 there may be problems of marker loss or loose linkage;

[0006] (3) High cost of breeding application: Multiple rounds of population screening and exchange of individual plants are required to verify the effectiveness of the markers, which significantly increases the breeding cycle and manpower costs.

[0007] II. Although comparative document 2 (CN119859638A discloses a maize plant architecture regulating gene ZmEXO1 and its application) reveals the regulatory role of the ZmEXO1 gene on plant architecture through gene editing technology, its technical approach has the following problems:

[0008] (1) High technical threshold: It relies on gene editing tools such as CRISPR / Cas9, which are complex to operate and require the construction of transgenic systems, and face challenges in terms of regulatory restrictions and public acceptance;

[0009] (2) Risk of functional redundancy: Gene editing may lead to non-target effects, such as mutations in ZmEXO1 may trigger cascaded variations in other agronomic traits, requiring additional verification of phenotypic stability;

[0010] (3) Limited scope of application: It is mainly for the verification of single gene function and is difficult to be directly applied to the improvement of complex plant height traits regulated by multiple genes.

[0011] III. 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 shortcomings:

[0012] (1) Low marker development efficiency: Candidate sites need to be screened through genome-wide association analysis (GWAS) or QTL mapping, the development cycle is long and depends on large-scale population data;

[0013] (2) Phenotypic association is unclear: The genetic correlation between some SNP markers and plant height has not been verified, resulting in insufficient accuracy of marker-assisted selection;

[0014] (3) Poor universality of detection system: primer design and reaction conditions lack standardization, and the comparability of results between different laboratories is low. Summary of the Invention

[0015] The purpose of this invention is to provide a Kasp marker PH-03-KASP-106 closely linked to maize plant height and its application, in order to solve the problems mentioned in the background art.

[0016] To achieve the above objectives, the present invention provides a Kasp marker PH-03-KASP-106 that is closely linked to maize plant height, including a single nucleotide polymorphism (SNP) site closely linked to the maize plant height trait and a KASP molecular marker primer set.

[0017] The single nucleotide polymorphism (SNP) site is located at position 163964821 on chromosome 3 (Chr3) of maize. Its polymorphism is expressed as T / C, in which the C allele is associated with the dwarf maize trait and the T allele is associated with the tall maize trait.

[0018] 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:

[0019] Forward primer 1: SEQ ID NO:1 (PH-03-KASP-106F1)

[0020] Forward primer 2: SEQ ID NO:2 (PH-03-KASP-106F2)

[0021] Reverse primer: SEQ ID NO:3 (PH-03-KASP-106R).

[0022] Preferably, the amplification region of the KASP molecular marker primer set covers the Chr3:163964621-163965021 interval, and the TT dwarf genotype and CC tall genotype are distinguished by the difference in fluorescence signal.

[0023] Preferably, the reagent for detecting maize plant height includes a KASP molecular marker primer set, a 2×Probe Mix A solution, and ddH2O, wherein the KASP molecular marker primer set comprises:

[0024] The concentrations of forward primer 1 and forward primer 2 are independently 4-10 μmol / L;

[0025] The concentration of the reverse primer is 4-10 μmol / L.

[0026] As a preferred method, detecting corn plant height specifically includes the following steps:

[0027] The maize genomic DNA was amplified by PCR using the KASP molecular marker primer set, and the fluorescence signal was detected by KASP genotyping. The plant height trait was determined according to the following rules:

[0028] TT genotype: orange-red fluorescent signal, corresponding to the dwarf trait;

[0029] CC genotype: blue fluorescent signal, corresponding to tall stem trait;

[0030] CT genotype: heterozygous signal.

[0031] Preferably, the PCR amplification reaction system is 10 μL, comprising:

[0032] Maize genomic DNA, 1-3 μL, at a concentration of 50-100 ng / μL;

[0033] KASP molecular marker primer set 0.1-0.15 μL;

[0034] 4-6 μL of 2×ProbeMixA solution;

[0035] Add ddH2O to a final volume of 10 μL.

[0036] Preferably, the ratio of forward primer 1: forward primer 2: reverse primer in the KASP molecular marker primer set is 2:2:5.

[0037] Preferably, the PCR amplification step is as follows:

[0038] Pre-denaturation at 95℃ for 10 minutes;

[0039] Denaturation at 95℃ for 20 seconds, annealing at 61℃ for 40 seconds, for a total of 10 cycles;

[0040] Denaturation at 95℃ for 20 seconds, annealing at 55℃ for 40 seconds, for a total of 31 cycles;

[0041] Keep at 25℃ for 10 minutes.

[0042] On the other hand, the present invention also provides an application of the Kasp marker PH-03-KASP-106 in maize breeding, for early identification, screening or molecular marker-assisted breeding of maize plant height traits, so as to improve the breeding efficiency of dwarf maize germplasm and for screening / preparing dwarf maize inbred lines.

[0043] Preferably, the method for preparing dwarf maize inbred lines is to use single nucleotide polymorphism (SNP) sites or the KASP marker PH-03-KASP-106 to selectively breed maize inbred lines with the TT genotype.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] (1) Improve breeding efficiency: With the help of the Kasp marker PH-03-KASP-106, maize plant height can be accurately identified in the early stage of maize breeding based on specific SNP sites and primer sets, and dwarf maize germplasm can be quickly screened, which can greatly shorten the breeding cycle, improve the breeding efficiency of dwarf maize germplasm, and accelerate the genetic selection and improvement process of maize varieties.

[0046] (2) Enhanced selection accuracy: The marker can accurately distinguish between TT dwarf, CC tall and CT heterozygous genotypes based on the difference in fluorescence signal. Breeders can accurately select target genotype plants based on the test results, effectively avoiding the error of traditional breeding that relies on phenotypic selection, enhancing selection accuracy and improving breeding success rate.

[0047] (3) Clarify the direction of molecular marker-assisted breeding: provide a basis for utilizing superior allelic variations related to maize plant height. In molecular marker-assisted breeding, help breeders to carry out more targeted hybridization, backcrossing and other operations based on molecular marker information, so as to achieve targeted breeding of maize plant height traits. For example, targeted breeding of TT genotype maize inbred lines to prepare dwarf maize inbred lines, thereby improving the scientific nature and operability of breeding work.

[0048] (4) Simple operation and cost-effectiveness: The detection method is relatively simple to operate, requiring only DNA extraction, PCR amplification and genotyping detection. At the same time, the parameters such as primer concentration, reaction system and amplification steps are clear, which is easy to standardize and apply on a large scale. It can reduce detection costs and improve detection efficiency while ensuring detection accuracy, and is suitable for maize breeding research and production practices of different scales. Attached Figure Description

[0049] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are explained in detail together with the embodiments of the invention, but do not constitute a limitation thereof.

[0050] Figure 1 This is a normal distribution diagram of plant height.

[0051] Figure 2 A distribution map of chromosomes marked with SNPs;

[0052] Figure 3 QQ plot and Manhattan plot for genome-wide association analysis of maize plant height;

[0053] Figure 4 Analysis of allelic variation effects at two important SNP loci related to plant height;

[0054] Figure 5 This is a partial result of the Sanger sequencing alignment of 300 candidate maize genes;

[0055] Figure 6 KASP molecular marker typing results for maize plant height in 256 maize accessions;

[0056] Figure 7 Analysis of the plant height effect value for KASP haplotypes;

[0057] Figure 8 Table 1 shows the KASP molecular marker scanning results for 256 maize plant heights.

[0058] Figure 9 Table 1 shows the KASP molecular marker scanning results for 256 maize plant heights.

[0059] Figure 10 This is a table showing the statistical results of KASP genotyping and phenotype. Detailed Implementation

[0060] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0061] The SNP locus provided by this invention is located at position 163964821 on chromosome 3 of maize, with 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 locus can be used to distinguish between tall and short maize plants.

[0062] This invention uses a KASP molecular marker primer set to select maize plant height traits. The identification of samples can be completed simply by DNA extraction, PCR-specific amplification, and KASP genotyping detection, resulting in dwarf maize. This molecular marker is effective in identifying maize plant height. Using the molecular marker-assisted selection of this invention can improve the efficiency of dwarf maize germplasm breeding, provide a basis for utilizing superior allelic variations related to maize plant height, and accelerate the breeding process.

[0063] This invention provides 1149 phenotypically diverse and representative DH lines selected from 5572 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 line was planted in two rows, 2.5 m long. The 21 main agronomic and yield traits were investigated according to standards, and data on traits such as plant height were recorded and compiled. After simplified genome resequencing of each line, the genome was assembled against a reference genome to obtain SNP polymorphic markers. GWAS association analysis was performed on the plant height trait data to obtain associated major SNPs, and haplotype analysis was conducted on the major SNPs. Based on field phenotypes, 150 maize inbred lines of each type (tall and short) were selected. The major SNP polymorphisms were verified by Sanger sequencing and the KASP markers in the sequences were obtained. KASP primers were designed based on the SNPs, and the plant height haplotypes of maize inbred lines were distinguished by gel-free fluorescent polymerase chain reaction.

[0064] This invention relates to specific primers that can effectively genotype maize plant height and detect differences in SNP polymorphisms. The KSAP marker for maize plant height provided by this invention can be used for marker-assisted breeding of maize plant height, which has important theoretical and practical guiding significance for accelerating the genetic selection and improvement of maize varieties.

[0065] The purpose of this invention is to locate SNP loci related to maize plant height and develop KASP-specific markers for identifying maize plant height based on locus sequence information. These molecular markers can predict maize plant height, providing molecular-assisted technology support for early identification and screening breeding of maize plant height traits.

[0066] Example 1: Investigation and Phenotypic Data Analysis of Plant Height Trait in Maize Inbred Lines

[0067] Using 1149 high-quality maize inbred lines bred over the past 15 years from the Shenzhen Genomics Institute of the Chinese Academy of Agricultural Sciences, these lines were grown under field conditions in Acheng District, Harbin City, Heilongjiang Province in 2023 and 2024. A randomized block design was employed, with each variety planted in two rows, each 3m long, with a row spacing of 0.65m and a plant spacing of 0.2m. Fertilization and irrigation were managed as usual in conventional field settings. Plant height was measured at maturity, excluding the first plant in each row. Five representative plants from each variety were selected to measure the distance from the ground to the top of the plant. Data on plant height and other traits were recorded and compiled. Statistical analysis of the phenotypic data under the two environments was performed using Microsoft Excel 2022 and IBM SPSS Statistics V27.0. The normality of the distribution was evaluated based on the coefficient of variation, skewness, and kurtosis. Finally, a frequency distribution histogram was plotted using Origin 2021 software to test the normality of the phenotypic data. Statistical analysis of maize plant height showed that the mean height ranged from 216.66 to 218.46 cm under both environmental conditions, with phenotypic variation ranging from 127.00 to 317.00 cm and a coefficient of variation ranging from 11.95% to 14.14%. The coefficient of variation exceeded 10% in all environmental conditions, indicating relatively rich phenotypic variation in plant height among the maize inbred lines. The absolute values ​​of skewness and kurtosis for plant height were both less than 1, and the data distribution curve conformed to a normal distribution, indicating that the plant height data conformed to quantitative trait characteristics (such as...). Figure 1 ), Figure 1 In the middle, a, b, and c represent respectively Figure 1 Plant height distribution maps for 2023, 2024, and BLUP. PH-2023H, PH-2024H, and PH-BLUP represent... Figure 1 Phenotypic data of maize plant height in Harbin in 2023 and 2024, and field phenotypic data of maize plant height under BLUP. The phenotypic data of maize plant height were analyzed using the 1me4 package in R language, and the generalized heritability was estimated by considering the variance of various influencing factors. The heritability of plant height was 86%, indicating that it is mainly influenced by genetic factors.

[0068] Example 2: Maize genomic DNA extraction, library construction, and sequencing

[0069] The specific method for constructing a library for the maize inbred lines in Example 1 is as follows:

[0070] (1) Weigh 1.0g of fresh leaves, cut them into small pieces and put them into a mortar. Grind them with liquid nitrogen and then add 3mL of 1.5×CTAB. Grind them into a homogenate and transfer it into a 15mL centrifuge tube. Then add 1mL of 1.5×CTAB to the mortar to rinse and transfer it into the centrifuge tube. Mix well and incubate in a 65℃ water bath for 30min, shaking slowly from time to time.

[0071] The 1.5×CTAB formulation is as follows (1L):

[0072]

[0073] Add deionized water to a final volume of 1L, and add mercaptoethanol to a final concentration of 0.2% (2ml) before use.

[0074] (2) After cooling to room temperature, add an equal volume of chloroform / isoamyl alcohol (24:1), mix gently until the lower layer turns dark green.

[0075] (3) Centrifuge at 4200 rpm for 10 min, 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 min. Incubate at -20℃ for 30 min to precipitate DNA.

[0076] (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.

[0077] (5) Detect the concentration of DNA and adjust it with water to 20 ng / ul.

[0078] (6) Database construction using the FBI-seq method (Zhao et al., 2023)

[0079] Example 3: GWAS analysis of maize plant height to obtain significant SNPs and candidate genes

[0080] All sequencing data were processed and analyzed using a high-performance computer server. Raw data processing: After quality assessment of the raw PE (Pair-end) sequencing data using FastQC, BWA was used for quality control. Sequencing reads were aligned to a reference genome (B73v4), and SNP detection was performed using GATK. After quality control filtering at the sample and variant levels, 57,849 high-quality SNP markers (minimum allele frequency >0.05, missing data <20%) were selected to ensure the accuracy and reliability of the analysis results. To better understand population structure and genetic background, a phylogenetic tree was constructed using iqTree software, principal component analysis (PCA) was performed on the whole genome SNP data using Plink software, and population structure analysis was performed using Faststructure software to clarify the genetic structure within the population. Genome-wide association analysis was performed on plant height and its BLUP value using the previously selected high-quality SNPs. A mixed linear model of genotype + phenotype + population structure + phylogenetic relationship matrix in GEMMA was used to analyze the association between SNP markers and various traits. All SNPs satisfying p < 1.7286e-5 were extracted from the GWAS results file using awk and converted to BED format files (Chr, Start, End). Two major-effect SNPs were obtained from GWAS analysis of plant height data over two years. The bedtoolsintersect tool was used to compare 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 [link to results]. Figures 2-4 , Figure 3 a and b are the QQ and Manhattan diagrams of plant height in 2023; Figure 3 .c and d are the QQ and Manhattan plots of plant height in 2024; Figure 3 .e and f are the QQ plot and Manhattan plot of plant height under BLUP conditions. 2023PH, 2024PH, and BLUP PH represent the plant height in 2023, 2024, and under BLUP conditions, respectively.

[0081] Example 4: Association analysis of candidate genes for maize plant height and discovery of new SNP sites and KASP markers

[0082] Based on field phenotypes, 150 maize inbred lines of each of the tall and short-stemmed types, and the maize B73 variety were selected. Genomic DNA was extracted from leaves using the CTAB method. The full-length sequences of candidate genes (including the 5' UTR region, 3' UTR, and all exon sequences) were amplified and Sanger sequenced for haplotype analysis. A segmented amplification method was used for DNA sequence amplification. Primers were designed using the free online primer design tool NCBI and synthesized by Shenzhen Sangon Biotech Co., Ltd. The amplification and sequencing primers are as follows:

[0083]

[0084] The sequences obtained from sequencing were submitted to Snapgene software for multiple sequence alignment. SNP sites with a deletion value greater than 20% and a secondary allele frequency (MAF) ≤ 5% were removed, verifying the authenticity of the SNP variant sites. A new SNP marker was also discovered, located at position 163964821 on maize chromosome 3. Single-gene association analysis was then performed using Tassel software, and haplotype results in NEX format containing only polymorphic sites (including indels) were output using Haploview software, along with LDblock plots. The distribution of each Haplotype in different subpopulations was statistically analyzed using Excel software, and the data was visualized using Origin software. The results are shown below. Figure 4 and Figure 5 , Figure 4 a and Figure 4 b represents the allelic variation effect analysis of two important SNP loci. 2023, 2024, and BLUP represent plant height in 2023, 2024, and under BLUP conditions, respectively. Figure 5 The Sanger sequencing comparison results of 300 maize candidate genes showed that the gene at Chr3:163964821 in dwarf maize was T, while the gene at Chr3:163964821 in tall maize was C.

[0085] Example 5: Development and Validation of KASP Markers for Maize Plant Height

[0086] Based on candidate gene association analysis, SNP variation sites that can be used to distinguish the height trait of different maize inbred lines were screened. KASP molecular markers capable of rapidly identifying two haplotypes were developed for these sites.

[0087] Specifically as follows:

[0088] (1) DNA was extracted from 249 maize inbred lines sequenced by Sanger sequencing using the CTAB method;

[0089] (2) Using DNA as a template, fluorescent polymerase chain reaction (PCR) genotyping was performed using KASP primers. The KASP primers were designed based on the SNP at position 163964821 on chromosome 3 of maize. The primers are shown in the table below:

[0090]

[0091] The PCR amplification reaction system, in 10 μl increments, consisted of: 2 μl of 4-50 ng / μl genomic DNA, 0.14 μl of primer mix (prepared by mixing 6 μl of forward primer 1, 6 μl of forward primer 2, 15 μl of reverse primer, and 23 μl of ddH2O), 5 μl of 2x ProbeMix A solution, and 3 μl of ddH2O.

[0092] The PCR amplification program was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s, 61℃ annealing for 40 s, 10 cycles; 95℃ denaturation for 20 s, 55℃ annealing for 40 s, 31 cycles; 25℃ for 10 min; 4℃ for storage.

[0093] After amplification, KASP detection was performed based on the AQP genotyping system operating instructions. The PCR program on the ABI 7500 qPCR instrument was set to 35℃ for 30 seconds. The results file was exported, and the genotypes were further determined according to the sample clusters. Results analysis was performed using Taqman Genotyper Software. The fluorescence values ​​corresponding to HEX and FAM for each PCR reaction well were obtained and divided by the value of the reference dye (ROX) for that well. The fluorescence values ​​were standardized to obtain the relative fluorescence values ​​of HEX and FAM for each PCR reaction well (FAM fluorescent tag sequences were observed at excitation wavelength of 485nm and emission wavelength of 520nm, and HEX fluorescent tag sequences were observed at excitation wavelength of 528nm and emission wavelength of 560nm). Based on the relative fluorescence values, the samples were clustered. The detection results are as follows: Figure 6 As shown.

[0094] Depend on Figure 6 It can be seen that there are two genotypes at this locus. 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 genotyping. The results showed that KASP markers can distinguish the two genotypes at Chr3:163964821.

[0095] Figure 6 The orange-red color represents TT, the blue color represents CC, and the gray color represents NTC as a control.

[0096] The analysis of the plant height trait of maize materials in Example 4 and the consistency between the typological results are as follows: Figure 8 , Figure 10 and Figure 2 As shown.

[0097] according to Figure 8 , Figure 10 and Figure 6 , Figure 7 It can be seen that when the fluorescence signal of the amplification product is orange-red, the maize plant height trait is identified as dwarf, corresponding to the TT genotype; when the fluorescence signal of the amplification product is blue, the maize plant height trait is identified as tall homozygous, i.e., CC. The KASP experiment results in Example 5 are consistent with the actual plant height traits of the tested samples, detecting 145 T / T materials (orange-red dots) and 111 C / C materials (blue dots). Among them, the tall trait and the dwarf trait have a 100% consistency with the actual traits of the tested materials, and the KASP experiment results are consistent with the sequencing results. Overall, in 256 samples tested, the KASP detection results and the sequencing results have a 100% consistency. This indicates that using this molecular marker to perform KASP experiments on the tested materials can effectively detect their genotypes, thereby completing the identification of germplasm.

[0098] This invention utilizes a specific Kasp marker, PH-03-KASP-106, for maize plant height detection, demonstrating significant benefits in various aspects, including maize breeding. The specific benefits are as follows:

[0099] (1) Improve breeding efficiency: With the help of the Kasp marker PH-03-KASP-106, maize plant height can be accurately identified in the early stage of maize breeding based on specific SNP sites and primer sets, and dwarf maize germplasm can be quickly screened, which can greatly shorten the breeding cycle, improve the breeding efficiency of dwarf maize germplasm, and accelerate the genetic selection and improvement process of maize varieties.

[0100] (2) Enhanced selection accuracy: The marker can accurately distinguish between TT dwarf, CC tall and CT heterozygous genotypes based on the difference in fluorescence signal. Breeders can accurately select target genotype plants based on the test results, effectively avoiding the error of traditional breeding that relies on phenotypic selection, enhancing selection accuracy and improving breeding success rate.

[0101] (3) Clarify the direction of molecular marker-assisted breeding: provide a basis for utilizing superior allelic variations related to maize plant height. In molecular marker-assisted breeding, help breeders to carry out more targeted hybridization, backcrossing and other operations based on molecular marker information, so as to achieve targeted breeding of maize plant height traits. For example, targeted breeding of TT genotype maize inbred lines to prepare dwarf maize inbred lines, thereby improving the scientific nature and operability of breeding work.

[0102] (4) Simple operation and cost-effectiveness: The detection method is relatively simple to operate, requiring only DNA extraction, PCR amplification and genotyping detection. At the same time, the parameters such as primer concentration, reaction system and amplification steps are clear, which is easy to standardize and apply on a large scale. It can reduce detection costs and improve detection efficiency while ensuring detection accuracy, and is suitable for maize breeding research and production practices of different scales.

[0103] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. The application of a KASP molecular marker primer set closely linked to maize plant height in the early identification or screening of maize plant height traits, characterized in that, The KASP molecular marker primer set includes: forward primer 1 as shown in SEQ ID NO:1, forward primer 2 as shown in SEQ ID NO:2, and reverse primer as shown in SEQ ID NO:3; maize with the TT genotype has a tall stem shape, and maize with the CC genotype has a short stem shape.

2. The application of a KASP molecular marker primer set closely linked to maize plant height in the breeding of tall maize, characterized in that, The KASP molecular marker primer set includes: forward primer 1 as shown in SEQ ID NO:1, forward primer 2 as shown in SEQ ID NO:2, and reverse primer as shown in SEQ ID NO:3; for the directional selection of maize with the TT genotype.

3. A method for detecting maize plant height, characterized in that, The process includes the following steps: PCR amplification of maize genomic DNA using the KASP molecular marker primer set described in claim 1, and detection of fluorescence signals by KASP genotyping. Plant height trait is determined according to the following rules: TT genotype: orange-red fluorescence signal, corresponding to dwarf trait; CC genotype: blue fluorescence signal, corresponding to tall trait; CT genotype: heterozygous signal. The ratio of forward primer 1: forward primer 2: reverse primer in the KASP molecular marker primer set is 2:2:

5.

4. The method for detecting maize plant height according to claim 3, characterized in that, The reagents used to detect maize plant height include a KASP molecular marker primer set, 2×ProbeMixA solution, and ddH2O. In the KASP molecular marker primer set, the concentrations of forward primer 1 and forward primer 2 are independently 4-10 μmol / L; the concentration of the reverse primer is 4-10 μmol / L.

5. The method for detecting maize plant height according to claim 4, characterized in that, The PCR amplification reaction system is 10 μL, which includes: 1-3 μL of maize genomic DNA at a concentration of 50-100 ng / μL; 0.1-0.15 μL of KASP molecular marker primer set; 4-6 μL of 2×ProbeMixA solution; and ddH2O to a final volume of 10 μL.

6. The method for detecting maize plant height according to claim 5, characterized in that, The PCR amplification steps are as follows: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 20 seconds, annealing at 61°C for 40 seconds, for a total of 10 cycles; denaturation at 95°C for 20 seconds, annealing at 55°C for 40 seconds, for a total of 31 cycles; and holding at 25°C for 10 minutes.

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