Indel marker ph-03-indel-106 associated with corn plant height trait and use thereof

Abstract

The present application relates to the technical field of plant molecular breeding, and particularly relates to an indel marker PH-03-Indel-106 related to the plant height trait of corn and application thereof. The indel marker is located in the region of the 3rd chromosome of corn Chr3:163962052-163964052, is a 7bp insertion / deletion polymorphism marker, the 7bp insertion sequence CTGAGGG exists in the tall inbred line of corn, and the insertion sequence does not exist in the short inbred line of corn. The specific primer pair PH-03-Indel-106F / R can specifically amplify the target region, and through 1.5% agarose gel electrophoresis detection, the 270bp band is determined as the short haplotype, and no band is the tall haplotype. The marker can be used for molecular marker assisted selection, breeding improvement and early identification and screening of the plant height trait of corn. The phenotype can be accurately predicted through DNA detection in the seedling stage, and environmental interference is avoided. The present application has the advantages of high specificity, simple detection, low cost, high throughput and the like, can significantly shorten the breeding period, realizes directional improvement of the plant height trait, and has important significance for genetic selection and plant type optimization of corn varieties.

Description

Technical Field

[0001] This invention relates to the field of plant molecular breeding technology, and more specifically, to the indel marker PH-03-Indel-106 related to maize plant height and its application. Background Technology

[0002] Maize plant height is an important agronomic trait affecting yield, lodging resistance, and mechanized harvesting. Improving plant height through marker-assisted selection (MAS) and gene editing is currently a research hotspot in maize breeding. Existing technologies for improving maize plant height mainly rely on gene editing or single nucleotide polymorphism (SNP) markers, but these methods have certain limitations:

[0003] I. Limitations of Gene Editing Technology

[0004] Chinese invention patent CN115927441B discloses a method for reducing maize plant height by mutating the AFB1 gene using CRISPR / Cas9 technology. While this method effectively creates dwarf plants by inhibiting AFB1 protein expression through gene editing, it has the following drawbacks:

[0005] Limitations of the application of genetically modified technology: CRISPR / Cas9 technology requires the introduction of exogenous gene vectors, which faces strict biosafety regulations and public acceptance issues in some regions, limiting its large-scale application in conventional breeding.

[0006] Operational complexity and cost: The process of constructing gene editing vectors, genetic transformation, and mutant screening is cumbersome, time-consuming, and requires high-level laboratory equipment and technology, which is not conducive to efficient breeding.

[0007] Genetic background dependence: The phenotypic effect of mutants may be affected by genetic background, requiring multiple backcrosses for purification and increasing the breeding cycle.

[0008] II. Limitations of SNP marker detection

[0009] Chinese invention patent CN102373278A discloses a SNP locus (SNP5259 in exon 5 of the Br2 gene) associated with maize plant height and its detection primers. This marker assists in plant height screening by detecting single-base variations, but it has the following problems:

[0010] The detection technology is complex: SNP labeling relies on technologies such as sequencing or quantitative PCR, which have high requirements for instruments and reagents and are not suitable for large-scale high-throughput screening.

[0011] Insufficient marker frequency and universality: For example, SNP5259 has a low frequency in natural populations (it exists only in a few inbred lines), and the detection results are limited by specific genetic backgrounds, making it difficult to apply widely to different germplasm resources.

[0012] Phenotypic prediction efficiency is limited: the effect of single-base variation may be affected by environmental factors, requiring multiple phenotypic verification, which increases breeding costs.

[0013] III. Common Problems of Existing Technologies

[0014] Insufficient early identification methods: Existing methods mostly rely on phenotypic surveys at maturity or complex molecular detection, lacking simple and efficient early plant height identification techniques, making it difficult to quickly screen target materials during the seedling stage.

[0015] Long breeding cycle: Whether it is gene editing or traditional molecular marker-assisted selection, multiple generations of self-crossing or backcrossing are required, resulting in a lengthy breeding cycle that is difficult to meet the needs of rapid breeding. Summary of the Invention

[0016] The purpose of this invention is to provide the indel marker PH-03-Indel-106 related to maize plant height and its application, in order to solve the problems mentioned in the background art.

[0017] To achieve the above objectives, this invention provides an indel marker PH-03-Indel-106 associated with maize plant height. The indel marker is located in the region of Chr3:163962052-163964052 on maize chromosome 3 and is a 7bp insertion / deletion polymorphic marker. Tall maize inbred lines have the 7bp insertion sequence CTGAGGG at this site, while dwarf maize inbred lines do not have the above insertion sequence at this site.

[0018] Preferably, the specific primer pair used to detect the Indel label has the following nucleotide sequence:

[0019] Forward primer PH-03-Indel-106F: 5'-CACCGAGGTCGGGACTAAAAAG-3';

[0020] Reverse primer PH-03-Indel-106R: 5'-CGATCAGCCTCAGACATGACC-3'.

[0021] Preferably, the method for detecting the Indel marker PH-03-Indel-106 includes the following steps:

[0022] (1) Extract maize genomic DNA;

[0023] (2) Perform PCR amplification using the specific primer pair described above;

[0024] (3) The PCR amplification products were detected by 1.5% agarose gel electrophoresis: if a specific band of 270bp appeared, it was identified as the dwarf haplotype Hap2; if the band did not appear, it was identified as the tall haplotype Hap1.

[0025] Preferably, maize inbred lines with the target plant height trait are screened by detecting the insertion / deletion status of the Indel marker in the target maize material: no insertion sequence corresponds to a short stalk phenotype: plant height <190cm, and the presence of an insertion sequence corresponds to a tall stalk phenotype: plant height >223cm.

[0026] Preferably, the specific primer pair specifically amplifies the region where the Indel marker PH-03-Indel-106 is located, and the tall / short haplotypes are distinguished by differences in electrophoretic bands.

[0027] Preferably, the PCR amplification reaction system contains 10 μL of Mix, 1 μL of DNA template, 0.5 μL of forward primer, 0.5 μL of reverse primer, and 8 μL of ddH2O; the reaction program is: 94℃ pre-denaturation for 30 seconds, 34 cycles, and a final extension at 72℃ for 5 minutes.

[0028] As a preferred option, each cycle consists of: denaturation at 98°C for 10 seconds, annealing at 62°C for 15 seconds, and extension at 72°C for 10 seconds.

[0029] On the other hand, the present invention provides an application of the indel marker PH-03-Indel-106 related to maize plant height traits, for molecular marker-assisted selection of maize plant height traits, for breeding to improve maize plant height traits, and for early identification and screening of maize plant height traits.

[0030] Preferably, the breeding for improving maize plant height specifically includes the following steps:

[0031] (1) Using the detection method of Indel marker PH-03-Indel-106, maize inbred lines with target plant height haplotype were screened as parents;

[0032] (2) Segregating populations were constructed through hybridization and backcrossing breeding. Genotype screening of offspring plants was carried out using the detection method of Indel marker PH-03-Indel-106, and new maize varieties with improved plant height were oriented to be bred.

[0033] Preferably, the early identification and screening of maize plant height trait specifically includes the following steps: collecting maize leaf samples at the seedling or maturity stage, performing PCR amplification using the specific primers, and predicting the plant height phenotype by detecting the Indel label PH-03-Indel-106.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] 1. Highly efficient and accurate phenotypic prediction

[0036] High specificity: The primer pair (PH-03-Indel-106F / R) can specifically amplify the target region and directly distinguish between the tall haplotype (Hap1, with the inserted sequence CTGAGGGG) and the short haplotype (Hap2, without the insertion) by the difference in electrophoretic bands (presence or absence of 270bp), avoiding interference from non-specific amplification.

[0037] The phenotypic correlation is clear: the insertion and deletion states correspond strictly to the plant height phenotype (dwarf plant height <190cm, tall plant height >223cm), and the phenotype can be predicted directly through molecular detection without relying on traditional phenotypic measurements.

[0038] 2. Significantly shortens the breeding cycle

[0039] Early screening: Genotyping can be performed by extracting DNA from leaves during the seedling stage, eliminating the need to wait for phenotypic observation at maturity, thus significantly shortening the breeding cycle and reducing time costs.

[0040] Precision-oriented breeding: By selecting inbred lines carrying target haplotypes (such as tall Hap1 or short Hap2) as parents, and combining backcross breeding and offspring genotype screening, rapid and targeted improvement of plant height traits can be achieved.

[0041] 3. Low-cost, high-throughput detection methods

[0042] Simplified experimental procedure: PCR reaction system design optimized (10μL system, only 3-step cycling conditions), combined with ordinary agarose gel electrophoresis (1.5%), no complicated equipment required, suitable for large-scale application in laboratories and fields.

[0043] Cost-effective: Compared to traditional phenotypic selection or SNP marker detection, Indel marker genotyping is less expensive, and electrophoresis can process samples in batches, improving screening efficiency.

[0044] 4. Improve the accuracy of breeding selection

[0045] Genetic markers are tightly linked: the markers are located in the Chr3:163962052-163964052 region on chromosome 3 and are highly associated with plant height, reducing environmental interference errors in traditional phenotypic selection.

[0046] Avoiding false positives: Specific primer design ensures reliable amplification results, and combined with clear insertion / deletion interpretation criteria (absence or absence of bands), the risk of misjudgment is significantly reduced.

[0047] 5. Wide applicability

[0048] Multi-scenario applications: It can be used in breeding programs for molecular marker-assisted selection (MAS), plant height trait improvement, and early seedling / maturity plant height identification, covering the needs of the entire breeding process.

[0049] Compatible with existing breeding systems: The method is simple and can be seamlessly integrated into the conventional maize breeding process without the need for additional technical training or equipment investment.

[0050] 6. Promote the optimization of maize varieties

[0051] Precise plant type regulation: By directionally improving plant height (such as tall-stalked, lodging-resistant varieties or short-stalked, densely planted varieties), the efficiency of light energy utilization, stress resistance, and yield potential are optimized, which helps to cultivate new high-yield and high-quality maize varieties.

[0052] In summary, this invention relates to specific primers that can effectively genotype maize plant height and detect differences in Indel polymorphisms; the Indel markers 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; the purpose of this invention is to locate the functional loci of maize plant height-related genes and develop Indel-specific markers for identifying maize plant height based on the locus sequence information; through these molecular markers, maize plant height can be predicted, providing molecular-assisted technical support for the early identification and screening breeding of maize plant height. Attached Figure Description

[0053] 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.

[0054] Figure 1 This is a normal distribution diagram of plant height.

[0055] Figure 2 A distribution map of chromosomes marked with SNPs;

[0056] Figure 3 QQ plot and Manhattan plot for genome-wide association analysis of maize plant height;

[0057] Figure 4 Analysis of allelic variation effects at two important SNP loci related to plant height;

[0058] Figure 5 This image shows a partial result of the Sanger sequencing alignment of 249 maize candidate genes.

[0059] Figure 6 Electrophoresis diagram of PCR products from internal reference primers of 200 maize inbred lines;

[0060] Figure 7Electrophoresis gel images of primer PCR products for plant height of 200 maize inbred lines;

[0061] Figure 8 Statistical graph of electrophoresis results of PCR products of maize endogenous gene IVR primers and plant height Indel molecular markers;

[0062] Figure 9 One of the statistical tables showing the results of polymer marker electrophoresis verification of the plant;

[0063] Figure 10 The second statistical table shows the results of the polymer marker electrophoresis verification of the plant.

[0064] Figure 11 The third figure shows the statistical table of the results of the molecular marker electrophoresis verification of the plant. Detailed Implementation

[0065] 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.

[0066] The indel marker PH-03-Indel-106, which is associated with maize plant height, is located in the region of Chr3:163962052-163964052 on maize chromosome 3. It is a 7bp insertion / deletion polymorphic marker. Tall maize inbred lines have the 7bp insertion sequence CTGAGGG at this site, while dwarf maize inbred lines do not have the above insertion sequence at this site.

[0067] 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. Major SNPs associated with plant height were obtained through association analysis, and haplotype analysis was performed on these major SNPs. Based on field phenotypes, 150 maize inbred lines of each type (tall, greater than 223 cm, and short, less than 190 cm) were selected. Major SNP polymorphisms were verified by Sanger sequencing and Indel markers in the sequences were obtained. PCR primers were designed based on the Indels, and plant height haplotypes of maize inbred lines were distinguished by PCR and agarose gel electrophoresis bands.

[0068] Example 1: Investigation and Phenotypic Data Analysis of Plant Height Trait in Maize Inbred Lines

[0069] 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 tip of the tassel. 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 1The phenotypic data of maize plant height were analyzed using the 1me4 package in R language, and the variance of various influencing factors was used to estimate the generalized heritability. The heritability of plant height was 86%, indicating that it is mainly affected by genetic factors.

[0070] Example 2: Maize genomic DNA extraction, library construction, and sequencing

[0071] The specific method for constructing a library for the maize inbred lines in Example 1 is as follows:

[0072] (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.

[0073] The 1.5×CTAB formulation is as follows (1L): 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 SNPs related to plant height trait were identified through GWAS analysis based on two years of plant height data, located within Chr3:163962052-163964052. The bedtools intersect tool was used to compare significant SNPs and their upstream and downstream 100 kb regions with the B73RefGen_v4 GFF gene annotation file to screen candidate genes. The results are shown in […]. Figure 2 and Figure 3 .

[0081] Example 4: Association analysis of candidate genes for maize plant height and mining of Indel markers

[0082] Based on field phenotypes, 150 maize inbred lines of each type (tall (greater than 223 cm) and short (less than 190 cm) and maize variety B73 were selected. Genomic DNA was extracted from leaves using the CTAB method. The full-length sequences of candidate genes (including the 5' UTR, 3' UTR, and all exon sequences) were amplified and sequenced using Sanger sequencing 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: 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 to verify the authenticity of the SNP variant sites. An indel marker was also identified. Single-gene association analysis was then performed using Tassel software. Haploview software was used to output haplotype results in NEX format containing only polymorphic sites (including indels), and LDblock plots were generated. Excel software was used to analyze the distribution of each haplotype in different subpopulations, and Origin software was used for data visualization. The results are shown below. Figure 4 and Figure 5 .

[0083] Example 5: Development and Validation of Indel Markers for Maize Plant Height

[0084] Based on candidate gene association analysis, Indel variation sites were screened to distinguish the height trait among different maize inbred lines. Molecular markers were then developed to rapidly identify the two haplotypes at these sites.

[0085] Specifically as follows:

[0086] (1) DNA was extracted from 249 maize inbred lines sequenced by Sanger sequencing using the CTAB method;

[0087] (2) Primer design and selection of internal reference primers:

[0088] Plant height Indel primer design: A 17-25 bp sequence was selected from the conserved region upstream of the Indel site as the upstream primer, and a downstream primer was designed at the Indel site. The Indel site is completely linked to the plant height trait; therefore, the presence or absence of PCR products can be initially determined based on whether the primers are linked to the maize plant height trait, and the accuracy of the molecular marker can be determined based on the actual results. The plant height primer design did not include the Indel; therefore, maize inbred lines without the Indel can produce PCR products, while maize inbred lines containing the Indel do not produce PCR products. Indel primers PH-03-Idel-106F: 5'-caccgaggtgggactaaaaag-3', PH-03-Idel-106R: 5'-CGATCAGCCTCAGACATGACC-3'; the internal control was selected using the universal maize primers IVR, with a product length of 226bp. IVR primers IVR-F: 5'-ccgctgtatcacaagggctggtacc-3', IVR-R: 5'-ggagcccgtgtagagcatgacgatc-3', as shown in the table below: (3) Perform PCR amplification using the designed primers. The PCR system is shown below: The PCR reaction program was as follows: 94℃ pre-denaturation for 30 seconds, 34 cycles of 98℃ denaturation for 10 seconds, 62℃ annealing for 15 seconds, 72℃ extension for 10 seconds, 72℃ extension for 5 minutes, and storage at 4℃.

[0089] (4) The presence or absence of amplification products for each material was detected by 1.5% agarose gel electrophoresis. The results are shown in […]. Figure 6 and Figure 7 .

[0090] (5) The presence or absence of the product is recorded based on the gel mapping, and the accuracy of the marking is then determined. The results are shown in the table below. Figure 9 , Figure 10 and Figure 11 .

[0091] Primers PH-03-Idel-106, designed based on the Indel variant site, amplified 200 maize inbred lines. PCR products with bands were recorded as C, those without bands as D, and the internal control primer band as B. Figure 6 and Figure 7 Analysis of the PCR product results revealed that all 200 maize inbred lines showed bands for the internal control primer IVR, indicating that the extracted maize DNA met the requirements for PCR. Figure 6 For the PH-03-Idel-106 primer, 77 out of 80 Hap2 maize inbred lines showed a 270bp band on molecular marker electrophoresis, while 3 showed no 270bp band. Similarly, 120 Hap1 inbred lines showed no 270bp band on molecular marker electrophoresis, resulting in a labeling accuracy of 96.25%. Figure 7 , Figure 9 , Figure 10 and Figure 11 ).

[0092] The beneficial effects of the indel marker PH-03-Indel-106 related to maize plant height and its application in this invention:

[0093] 1. Highly efficient and accurate phenotypic prediction

[0094] High specificity: The primer pair (PH-03-Indel-106F / R) can specifically amplify the target region and directly distinguish between the tall haplotype (Hap1, with the inserted sequence CTGAGGGG) and the short haplotype (Hap2, without the insertion) by the difference in electrophoretic bands (presence or absence of 270bp), avoiding interference from non-specific amplification.

[0095] The phenotypic correlation is clear: the insertion and deletion states correspond strictly to the plant height phenotype (dwarf plant height <190cm, tall plant height >223cm), and the phenotype can be predicted directly through molecular detection without relying on traditional phenotypic measurements.

[0096] 2. Significantly shortens the breeding cycle

[0097] Early screening: Genotyping can be performed by extracting DNA from leaves during the seedling stage, eliminating the need to wait for phenotypic observation at maturity, thus significantly shortening the breeding cycle and reducing time costs.

[0098] Precision-oriented breeding: By selecting inbred lines carrying target haplotypes (such as tall Hap1 or short Hap2) as parents, and combining backcross breeding and offspring genotype screening, rapid and targeted improvement of plant height traits can be achieved.

[0099] 3. Low-cost, high-throughput detection methods

[0100] Simplified experimental procedure: PCR reaction system design optimized (10μL system, only 3-step cycling conditions), combined with ordinary agarose gel electrophoresis (1.5%), no complicated equipment required, suitable for large-scale application in laboratories and fields.

[0101] Cost-effective: Compared to traditional phenotypic selection or SNP marker detection, Indel marker genotyping is less expensive, and electrophoresis can process samples in batches, improving screening efficiency.

[0102] 4. Improve the accuracy of breeding selection

[0103] Genetic markers are tightly linked: the markers are located in the Chr3:163962052-163964052 region on chromosome 3 and are highly associated with plant height, reducing environmental interference errors in traditional phenotypic selection.

[0104] Avoiding false positives: Specific primer design ensures reliable amplification results, and combined with clear insertion / deletion interpretation criteria (absence or absence of bands), the risk of misjudgment is significantly reduced.

[0105] 5. Wide applicability

[0106] Multi-scenario applications: It can be used in breeding programs for molecular marker-assisted selection (MAS), plant height trait improvement, and early seedling / maturity plant height identification, covering the needs of the entire breeding process.

[0107] Compatible with existing breeding systems: The method is simple and can be seamlessly integrated into the conventional maize breeding process without the need for additional technical training or equipment investment.

[0108] 6. Promote the optimization of maize varieties

[0109] Precise plant type regulation: By directionally improving plant height (such as tall-stalked, lodging-resistant varieties or short-stalked, densely planted varieties), the efficiency of light energy utilization, stress resistance, and yield potential are optimized, which helps to cultivate new high-yield and high-quality maize varieties.

[0110] This invention relates to specific primers that can effectively genotype maize plant height and detect differences in Indel polymorphism. The Indel markers 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.

[0111] The purpose of this invention is to locate the functional loci of maize plant height-related genes and develop Indel-specific markers for identifying maize plant height based on the 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.

[0112] 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 indel marker PH-03-Indel-106 associated with maize plant height, characterized in that, The specific primer pair for detecting the Indel label has the following nucleotide sequence: Forward primer PH-03-Indel-106F: 5'-CACCGAGGTCGGGACTAAAAAG-3'; Reverse primer PH-03-Indel-106R: 5'-CGATCAGCCTCAGACATGACC-3'; The Indel marker is located between positions 13 and 14 at the 5' end of the reverse primer, containing a 7bp insertion sequence CTGAGGG. Tall maize inbred lines have the 7bp insertion sequence CTGAGGG at this site, while dwarf maize inbred lines do not.

2. The indel marker PH-03-Indel-106 related to maize plant height trait according to claim 1, characterized in that, The method for detecting the Indel marker PH-03-Indel-106 includes the following steps: (1) Extract maize genomic DNA; (2) PCR amplification using the primer pair described in claim 1; (3) The PCR amplification products were detected by 1.5% agarose gel electrophoresis: if a specific band of 270bp appeared, it was identified as the dwarf haplotype Hap2; if the band did not appear, it was identified as the tall haplotype Hap1.

3. The indel marker PH-03-Indel-106 related to maize plant height trait according to claim 2, characterized in that, By detecting the insertion / deletion status of the Indel marker in the target maize material, maize inbred lines with the target plant height trait were screened: no insertion sequence corresponds to the short stalk phenotype: plant height <190cm, and the presence of the insertion sequence corresponds to the tall stalk phenotype: plant height >223cm.

4. The indel marker PH-03-Indel-106 related to maize plant height trait according to claim 1, characterized in that, The specific primer pair can specifically amplify the region where the Indel label PH-03-Indel-106 is located, and the tall / short haplotypes can be distinguished by the difference in electrophoretic bands.

5. The indel marker PH-03-Indel-106 related to maize plant height trait according to claim 2, characterized in that, The PCR amplification reaction system contained 10 μL of Mix, 1 μL of DNA template, 0.5 μL of forward primer, 0.5 μL of reverse primer, and 8 μL of ddH2O; the reaction program was: 94℃ pre-denaturation for 30 seconds, 34 cycles, and a final extension at 72℃ for 5 minutes.

6. The indel marker PH-03-Indel-106 related to maize plant height trait according to claim 5, characterized in that, Each cycle consists of: denaturation at 98°C for 10 seconds, annealing at 62°C for 15 seconds, and extension at 72°C for 10 seconds.

7. The application of a primer for detecting the indel marker PH-03-Indel-106, as described in any one of claims 1-6, which is associated with the maize plant height trait, characterized in that, Marker-assisted selection for maize plant height, breeding for improvement of maize plant height, and early identification and screening of maize plant height.

8. The application of the indel marker PH-03-Indel-106 related to maize plant height trait as described in claim 7, characterized in that, The breeding for improving maize plant height specifically includes the following steps: (1) Using the detection method of Indel marker PH-03-Indel-106, maize inbred lines with target plant height haplotype were screened as parents; (2) Segregating populations were constructed through hybridization and backcrossing breeding. Genotype screening of offspring plants was carried out using the detection method of Indel marker PH-03-Indel-106, and new maize varieties with improved plant height were oriented to be bred.

9. The application of the indel marker PH-03-Indel-106 related to maize plant height trait as described in claim 7, characterized in that, The early identification and screening of maize plant height trait specifically includes the following steps: collecting maize leaf samples at the seedling or maturity stage, performing PCR amplification using the specific primers, and predicting the plant height phenotype by detecting the Indel label PH-03-Indel-106.

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