A KASP molecular marker PH5-KASP based on a SNP site of a corn Zm00001eb259660 gene and application thereof

Abstract

This invention relates to the field of plant molecular breeding technology, specifically to a KASP molecular marker PH5-KASP based on the SNP site of the maize Zm00001eb259660 gene and its application. It provides a KASP molecular marker targeting the SNP site at 226029364 bp on maize chromosome 5, which is a functional missense mutation c.485C>T in the coding region of the Zm00001eb259660 gene. The marker consists of two allele-specific forward primers carrying different fluorescent tags and one universal reverse primer, stably distinguishing between C:C and T:T homozygous genotypes, where C:C corresponds to higher plant height and T:T corresponds to lower plant height. This invention offers high marker genotyping accuracy, simple and low-cost detection, and is suitable for large-scale breeding sample detection. It can be used for early identification of maize plant height traits, germplasm screening, and assisted breeding, significantly improving the efficiency of ideal maize plant architecture improvement.

Description

Technical Field

[0001] This invention relates to the field of plant molecular breeding technology, and more specifically, to a KASP molecular marker PH5-KASP based on the SNP site of the maize Zm00001eb259660 gene and its application. Background Technology

[0002] Maize is the most widely planted and highest-yielding grain crop in my country, and also an important feed and industrial processing raw material. Its yield performance, stress resistance, and adaptability directly affect national food security and the stability of the agricultural industry chain. Plant height is one of the core selective traits in maize breeding. It not only directly regulates the canopy structure, light energy utilization efficiency, and lodging resistance of the plant population, but also determines the variety's potential for dense planting and its suitability for mechanized harvesting. It is a core targeted trait for improving the ideal plant type of maize. Therefore, identifying key genetic variations significantly associated with maize plant height and developing efficient and precise molecular marker tools are of great practical significance for shortening the maize breeding cycle, improving the efficiency of target trait selection, and realizing molecular design breeding.

[0003] With the rapid development of high-throughput sequencing and molecular marker technologies, single nucleotide polymorphism (SNP) markers have become the mainstream marker type in crop genetic research and molecular breeding applications due to their advantages such as high density in the genome, strong genetic stability, and ease of automated high-throughput detection. Among them, competitive allele-specific PCR (KASP) technology, with its accurate genotyping results, simple detection process, low cost per sample, no need for gel electrophoresis, and suitability for large-scale batch detection of breeding samples, has gradually become the core technology in marker-assisted selection for crops. It is widely used in genotyping, target trait prospect selection, and germplasm resource genetic background analysis of major crops such as maize and rice.

[0004] In the prior art, Chinese invention patent CN118028529B discloses a KASP molecular marker for identifying maize stalk puncture strength and its application. This invention, through genome-wide association analysis of natural maize populations, locates SNP loci significantly associated with maize stalk puncture strength and develops corresponding KASP molecular markers based on these loci. These markers can be used for marker-assisted selection of maize stalk strength traits, providing an effective molecular tool for improving maize lodging-related stalk traits. However, this technology targets only maize stalk puncture strength and does not involve the mining of key genetic variations and the development of dedicated molecular markers for maize plant height traits. Therefore, it cannot be directly applied to genotyping and assisted breeding of maize plant height traits.

[0005] Currently, despite extensive genetic mapping studies on maize plant height both domestically and internationally, stable, efficient KASP molecular markers with clearly defined functional gene backgrounds that can be directly applied to commercial breeding practices remain very limited. First, many existing plant height-related molecular markers are derived from preliminary QTL mapping or association analysis results. The marker sites are often genetically distant from the functional causative mutations controlling plant height. When applied in breeding populations with different genetic backgrounds, genetic recombination can easily lead to marker segregation from the trait, resulting in insufficient accuracy and universality in marker selection. Second, most reported maize plant height-related SNP markers only demonstrate statistically significant associations, lacking clear candidate genes for support. The failure to locate functional variations in the gene coding region that can lead to protein sequence changes hinders precise molecular design breeding with clear mechanistic analysis. Finally, the detection of some plant height-related molecular markers relies on costly gene chips or whole-genome resequencing platforms, resulting in long detection cycles and high single-sample detection costs, failing to meet the needs for rapid, low-cost early screening in large-scale field breeding segregating populations.

[0006] In summary, developing dedicated KASP molecular markers that target functional variations in maize plant height, have a clear genetic background, and are easy and efficient to detect are urgent technical problems to be solved in the field of molecular breeding for ideal maize plant type. Summary of the Invention

[0007] The purpose of this invention is to provide a KASP molecular marker PH5-KASP based on the SNP site of the maize Zm00001eb259660 gene and its application, in order to solve the problems mentioned in the background art, such as the long genetic distance, low selection accuracy, high detection cost, and lack of clear functional gene support of existing maize plant height-related molecular markers.

[0008] To achieve the above objectives, this invention provides a KASP molecular marker PH5-KASP based on the SNP site of the maize Zm00001eb259660 gene. The KASP molecular marker specifically targets a single nucleotide polymorphism site at 226,029,364 bp on maize chromosome 5. The SNP site is located in the coding region of exon 1 of the T001 transcript of the maize Zm00001eb259660 gene and is a missense mutation of c.485C>T. The KASP molecular marker consists of a first allele-specific forward primer, a second allele-specific forward primer, and a universal reverse primer. The first allele-specific forward primer carries a FAM fluorescent tag sequence and specifically binds to the upstream flanking sequence of the C allele at the SNP site. The second allele-specific forward primer carries a HEX fluorescent tag sequence and specifically binds to the upstream flanking sequence of the T allele at the SNP site. The universal reverse primer binds to the conserved genomic sequence downstream of the SNP site.

[0009] Preferably, the nucleotide sequence of the first allele-specific forward primer is 5'-GAAGGTGACCAAGTTCATGCTTACATCGAATGCAGTTCAAAAAC-3', the nucleotide sequence of the second allele-specific forward primer is 5'-GAAGGTCGGAGTCAACGGATTTACATCGAATGCAGTTCAAAAAT-3', and the nucleotide sequence of the universal reverse primer is 5'-GCTGGGGCGACAGTACCT-3'.

[0010] Preferably, the SNP site is derived from the association analysis results of multi-point phenotypes and whole-genome resequencing of 377 maize inbred lines. It is located within a linkage disequilibrium range of 400kb upstream and downstream of the significant association interval for plant height trait, and is the only functional variant site in the coding region of candidate gene Zm00001eb259660 that is highly significantly associated with plant height trait.

[0011] Preferably, the c.485C>T missense mutation causes the 162nd amino acid residue of the protein encoded by the maize Zm00001eb259660 gene to be replaced by isoleucine.

[0012] The present invention also provides a genotypic identification kit for maize plant height including the above-mentioned KASP molecular marker PH5-KASP. The kit further includes PCR amplification reagent, fluorescence signal detection reagent, negative control and positive control. The PCR amplification reagent contains 2×ProbeMixA solution and enzyme-free water ddH2O. The KASP molecular marker PH5-KASP is aliquoted and stored in the form of premixed primer mix.

[0013] Preferably, the premixed primer Mix is ​​prepared by mixing 6 μl of first allele-specific forward primer, 6 μl of second allele-specific forward primer, 15 μl of universal reverse primer, and 23 μl of enzyme-free water ddH2O in a volume ratio; the negative control is template-free enzyme-free water ddH2O, and the positive control is maize genomic DNA of C:C homozygous genotype and T:T homozygous genotype verified by resequencing.

[0014] In addition, the above-mentioned KASP molecular marker PH5-KASP or maize plant height genotype identification kits are used in maize plant height-related genotype detection, maize germplasm resource plant height trait genotype survey, and maize molecular marker-assisted breeding.

[0015] Preferably, the application includes detecting the genotype of the maize material to be tested at the 226,029,364bp site on chromosome 5 of maize, and classifying the maize material to be tested into two haplotypes based on the detection results, wherein the C:C homozygous genotype corresponds to haplotype Hap1, and the T:T homozygous genotype corresponds to haplotype Hap2.

[0016] As a preferred method, the method for detecting the genotype of the maize material to be tested includes the following steps: S1: Genomic DNA was extracted from fresh leaves of maize seedlings using a modified CTAB method. After quality testing by agarose gel electrophoresis and UV spectrophotometer, the DNA concentration was uniformly adjusted to 4-50 ng / μl. S2: Using the genomic DNA extracted in step S1 as a template, PCR amplification reaction is performed using the KASP molecular marker PH5-KASP; S3: After the PCR amplification reaction was completed, the amplification products were incubated at 35℃ for 30s, and the FAM fluorescence signal and HEX fluorescence signal were collected respectively. The FAM fluorescence signal was detected at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescence signal was detected at an excitation wavelength of 528nm and an emission wavelength of 560nm. S4: The collected signals were standardized based on the fluorescence value of the reference dye ROX, and the genotype of the maize material to be tested was determined by cluster analysis.

[0017] Preferably, the PCR amplification reaction system in step S2, in a total volume of 10 μl, consists of: 2 μl of genomic DNA at a concentration of 4-50 ng / μl, 0.14 μl of premixed primer Mix, 5 μl of 2×ProbeMixA solution, and 3 μl of enzyme-free water ddH2O. The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s, 61℃ annealing for 40 s, for a total of 10 cycles; 95℃ denaturation for 20 s, 55℃ annealing for 40 s, for a total of 31 cycles; incubation at 25℃ for 10 min; and storage of the amplification products at 4℃ in the dark.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The SNP locus provided by this invention is directly located in the coding region of the maize gene Zm00001eb259660. It is a functional missense mutation that can lead to changes in the amino acid sequence of the encoded protein, rather than a genomic spacer variant indirectly linked to the trait. This eliminates the risk of segregation between the marker and the target trait due to genetic recombination from a genetic perspective. Compared to existing plant height-related markers obtained solely based on statistical association, the marker locus of this invention has zero genetic distance from the causative variation controlling plant height. When applied in breeding populations with different genetic backgrounds, there will be no decoupling between the marker and the trait, significantly improving selection accuracy. Furthermore, this locus has been validated through association between multiple field phenotypes and natural populations, showing a stable genetic effect on plant height under different planting environments. There are no effect fluctuations caused by environmental interactions, and it can stably distinguish phenotypic differences between different alleles, solving the technical problems of poor universality and unstable genetic effects of existing plant height markers.

[0019] 2. This invention utilizes a KASP molecular marker developed based on a target SNP locus. Primer design targets conserved genomic sequences flanking the locus, resulting in high amplification specificity and clear, definitive genotyping results. It can stably distinguish between two homozygous alleles, with genotyping results showing over 98% consistency with whole-genome resequencing, demonstrating high detection reliability. Detection of this marker can be completed using only a conventional quantitative real-time PCR platform, eliminating the need for costly gene chips or high-throughput sequencing platforms. The single-sample detection cycle is short and cost-effective, with a standardized procedure that eliminates the need for subsequent gel electrophoresis or sequencing steps. This makes it suitable for batch detection of large-scale segregating populations in breeding processes, solving the technical problems of high cost, complex procedures, and difficulty in applying existing plant height-related marker detection methods to field breeding practices.

[0020] 3. The molecular marker provided by this invention enables early genotypic selection of maize plant height, allowing genotypic identification of target loci at the seedling stage without waiting for physiological maturity for plant height phenotypic surveys, effectively shortening the breeding selection cycle. Simultaneously, this marker can be directly used for targeted molecular improvement of maize plant height. Through marker-assisted backcrossing, T alleles controlling lower plant height can be precisely introduced, reducing the workload of phenotypic identification during backcrossing, minimizing the interference of environmental factors on selection results, and improving the accuracy and efficiency of plant height trait improvement. Furthermore, this marker can be used for large-scale genotypic surveys of maize germplasm resources to rapidly identify germplasm materials carrying superior alleles, providing new technical support and material basis for breeding ideal maize plant types. Attached Figure Description

[0021] 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.

[0022] Figure 1 This is a normal distribution diagram of maize plant height trait in an embodiment of the present invention; Figure 2 This is a chromosome distribution diagram of SNP markers according to an embodiment of the present invention; Figure 3 The Manhattan plot and QQ plot are used for genome-wide association analysis of rice plant height in embodiments of the present invention. Figure 4 This is a diagram showing the LD attenuation distance in an embodiment of the present invention; Figure 5 This is a phenotypic association diagram of the Zm00001eb259660 gene haplotype in an embodiment of the present invention; Figure 6 This is a graph showing the KASP molecular marker typing results of this invention; Figure 7 This is a KASP typing and phenotypic effect diagram of the present invention. Detailed Implementation

[0023] 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention provides a KASP molecular marker PH5-KASP based on a SNP site in the maize Zm00001eb259660 gene and its application. The KASP molecular marker specifically targets a single nucleotide polymorphism (SNP) site at 226029364 bp on chromosome 5 of maize. This SNP site is located in the coding region of exon 1 of the T001 transcript of the maize Zm00001eb259660 gene, and is a missense mutation of c.485C>T, resulting in the replacement of threonine with isoleucine at amino acid position 162 of the protein encoded by the maize Zm00001eb259660 gene. The KASP molecular marker consists of a first allele-specific forward primer, a second allele-specific forward primer, and a universal reverse primer. The first allele-specific forward primer carries a FAM fluorescent tag sequence and specifically binds to the upstream flanking sequence of the C allele at the SNP site, with the nucleotide sequence 5'-GAAGGTGACCAAGTTCATGCTTACATCGAATGCAGTTCAAAAAC-3'. The second allele-specific forward primer carries a HEX fluorescent tag sequence and specifically binds to the upstream flanking sequence of the T allele at the SNP site, with the nucleotide sequence 5'-GAAGGTCGGAGTCAACGGATTTACATCGAATGCAGTTCAAAAAT-3'. The universal reverse primer binds to the conserved genomic sequence downstream of the SNP site, with the nucleotide sequence 5'-GCTGGGGCGACAGTACCT-3'. Example 1: Phenotypic Data Analysis of Maize Plant Height

[0025] Four hundred genetically rich maize inbred lines were selected for a field survey of plant height in two pilot areas in Shuangcheng and Daowai districts of Harbin, Heilongjiang Province. The Shuangcheng pilot area was located at 126°19′40.332″E, 45°24′23.816″N. The Daowai pilot area was located at 126°47.3604′E, 45°51.4494′N. A randomized block design was used in the field trials, with three biological replicates. Each variety was planted in two rows per replicate, with a row length of 3m, a row spacing of 0.65m, and a plant spacing of 0.2m. Fertilizer and water management followed local conventional field production standards, and pest, disease, and weed control were carried out normally throughout the growth period to ensure normal growth and development of the maize plants. Plant height was measured at the physiological maturity stage of maize. Plants at the two ends of each row were excluded. Five representative healthy plants were selected for each variety and each replicate. The vertical distance from the ground to the top of the tassel was measured and the original plant height data was recorded.

[0026] Raw plant height data from the two pilot sites were processed, and phenotypic data were statistically analyzed using Microsoft Excel 2024 and Rstudio V4.5.1 software. The mean, standard deviation, and coefficient of variation of the plant height data from both pilot sites were calculated. The mean plant height data from the Daowai pilot site was 213.48 cm, with a coefficient of variation of 12.25% and a standard deviation of 26.16 cm. The mean plant height data from the Shuangcheng pilot site was 194.31 cm, with a coefficient of variation of 12.90% and a standard deviation of 25.07 cm. The heritability of plant height was calculated using the restricted maximum likelihood method, yielding a heritability of 72%, indicating that this trait is mainly controlled by genetic factors and has high genetic stability, making it suitable for subsequent genetic mapping studies. Distribution curves were plotted for the plant height data from the two pilot sites and the calculated best linear unbiased predicted values. All three sets of data conformed to a normal distribution, indicating that plant height is a typical quantitative trait, meeting the trait requirements for genome-wide association analysis. Figure 1 As shown. Example 2: Maize genomic DNA extraction and resequencing library construction

[0027] For the 400 maize inbred lines in Example 1, fresh healthy leaves were taken from each material during the seedling stage, and genomic DNA was extracted using the modified CTAB method. The specific operation steps are as follows.

[0028] Weigh 1.0g of fresh leaves, cut them into small pieces, and place them in a pre-cooled mortar. Add liquid nitrogen and grind thoroughly until powdered. Add 3mL of 1.5 times the concentration of CTAB extract preheated to 65°C, and continue grinding into a homogenate. Transfer the homogenate to a 15mL centrifuge tube. Add another 1mL of 1.5 times the concentration of CTAB extract at the same temperature to the mortar to rinse away any remaining tissue. Transfer the rinse solution to the centrifuge tube as well. Gently invert the centrifuge tube to mix, and then place it in a 65°C water bath for 30 minutes. Gently invert the centrifuge tube every 10 minutes to ensure that the extract and tissue are in full contact.

[0029] The preparation method for a 1.5x concentration CTAB extract is as follows: Weigh 15g CTAB, measure 75mL of a 1mol / L Tris.Cl solution with a pH of 8.0, measure 30mL of a 0.5mol / L EDTA solution, weigh 61.4g NaCl, and add deionized water to a final volume of 1L. Before use, add 0.2% mercaptoethanol and mix thoroughly.

[0030] After the mixture in the centrifuge tube has cooled to room temperature, add an equal volume of chloroform and isoamyl alcohol mixture (chloroform to isoamyl alcohol volume ratio 24:1). Gently invert the centrifuge tube to mix for 10 minutes, until the lower organic phase turns dark green. Place the centrifuge tube in a centrifuge and centrifuge at 4200 rpm for 10 minutes. Carefully transfer the upper aqueous phase to a new 15 mL centrifuge tube, add 2 volumes of anhydrous ethanol pre-cooled to -20°C, gently invert to mix, and let stand for 5 minutes. Place in a -20°C freezer for 30 minutes to allow the genomic DNA to precipitate completely.

[0031] Remove the centrifuge tube and centrifuge at 4200 rpm for 10 min, discarding the supernatant. Add 1 mL of 75% ethanol to wash the precipitate once, centrifuge at 4200 rpm for 5 min, and discard the supernatant. Invert the centrifuge tube onto sterile absorbent paper and allow the DNA precipitate to dry at room temperature until no alcohol residue remains. Add 50 μL of TEL buffer to dissolve the DNA precipitate. Assess DNA integrity using 1% agarose gel electrophoresis and determine DNA concentration and purity using a UV spectrophotometer. Adjust the DNA concentration of all samples to a uniform 20 ng / μL and store at -20°C for later use.

[0032] A simplified AIO-seq method was used to construct genome resequencing libraries. Genomic DNA from each sample was fragmented using Tn5 transposase, with fragment lengths ranging from 300 bp to 500 bp. MGI sequencing-specific tag adapters were ligated into each sample during PCR amplification. After mixing equal volumes of amplification products from all samples, fragment size was screened using magnetic beads, and fragments approximately 400 bp in length were recovered. The selected mixed samples were circularized using the Hieff NGS Fast-Pace DNA circularization kit. The circularization products were quantified using the Qubitss DNA detection kit. Finally, the qualified circularized libraries were sequenced on an MGIDNBSEQ-T1 sequencer, generating 150 bp paired-end reads, yielding approximately 5 times the coverage depth of whole-genome resequencing data for each sample.

[0033] The 1.5×CTAB formulation is as follows (1L): Example 3: Genome-wide association analysis and candidate gene screening for maize plant height

[0034] For the raw sequencing data from the resequencing process, FastQC software was first used to assess the quality of the raw paired-end reads, removing low-quality reads and adapter contamination sequences. BWA software was then used to align the quality-controlled reads to the maize B73v5 reference genome, obtaining preliminary alignment results. GATK software was then used to detect genome-wide SNPs and Indels. The detected raw variants underwent quality control filtering at both the sample and variant levels, with the filtering criteria being a minimum allele frequency greater than or equal to 0.05 and a deletion rate less than or equal to 20%. Ultimately, 8,774,641 high-quality SNP markers and 1,463,328 Indel markers were obtained for subsequent analysis.

[0035] Principal component analysis (PCA) was performed on genome-wide SNP data using Plink software. The first three principal components were extracted and used as covariates for subsequent association analyses to control for the influence of population structure on the association results. The phylogenetic matrix between materials was calculated using GCTA software, and population structure analysis was performed using Admixture software to clarify the genetic background structure of the tested populations. Using high-quality SNP markers obtained from the initial screening, genome-wide association analyses were conducted on plant height trait data and the best unbiased linear predictor values ​​in GEMMA software using a mixed linear model. The phylogenetic matrix and the first three principal components were included as covariates during the analysis to reduce false positive association results.

[0036] From the genome-wide association analysis results, all significant SNPs satisfying a p-value less than 1.7286 x 10^-5 were extracted to obtain major-effect SNPs significantly associated with plant height. The linkage disequilibrium decay distance of the population was calculated using LDdecay software, yielding a distance of 400 kb. The upstream and downstream 400 kb linkage disequilibrium regions of the significant SNPs were screened, and the regions were compared with the gene annotation file of version B73RefGen_v5, extracting all annotated genes within these regions as candidate genes. Genes showing significant association in both pilot studies and the best linear unbiased prediction value, and exhibiting functional variations in the coding region, were prioritized for screening. Finally, Zm00001eb259660 located on chromosome 5 was identified as the final candidate gene.

[0037] All variant sites within the candidate gene Zm00001eb259660 were extracted, and a C / T variant site located at 226.029, 364 bp on chromosome 5 was identified. This site, located in the coding region, is a missense mutation, resulting in a change from threonine to isoleucine at amino acid position 162 of the encoded protein. Based on the genotype of this site, the tested population was divided into two main haplotypes: the C allele homozygous haplotype was named Hap1, and the T allele homozygous haplotype was named Hap2. Association analysis between the two haplotypes and the plant height phenotypes in the two pilot areas showed highly significant differences in plant height for both haplotypes in both environments, with consistent effect directions, indicating that this site has a stable genetic effect and can be used for subsequent molecular marker development, such as… Figures 2-5 As shown. Example 4: Development and Validation of KASP Markers for Maize Plant Height

[0038] Based on the key SNP sites screened in Example 3, a KASP molecular marker was developed and named PH5-KASP. Two allele-specific forward primers carrying different fluorescent tags were designed for the C and T alleles at this site, along with one universal reverse primer. The first allele-specific forward primer has a FAM fluorescent tag sequence attached to its 5' end, specifically recognizing the C allele. The second allele-specific forward primer has a HEX fluorescent tag sequence attached to its 5' end, specifically recognizing the T allele. The universal reverse primer binds to a conserved sequence downstream of the binding site.

[0039] KASP primers were designed based on the SNPs at positions 226, 029, and 364 on chromosome 5 of maize. The primers are shown in the table below:

[0040] Prepare a primer mixture for KASP marker detection by taking 6 μL of first allele-specific forward primer, 6 μL of second allele-specific forward primer, 15 μL of universal reverse primer, and 23 μL of enzyme-free water, mixing them thoroughly to obtain the primer mixture, and storing it at -20 degrees Celsius in the dark for later use.

[0041] A genotyping kit for maize plant height was prepared. The kit contained the primer mixture described above, a 2x concentration of ProbeMixA solution, enzyme-free water, a negative control, and a positive control. The negative control was template-free enzyme-free water. The positive control consisted of maize genomic DNA with C:C homozygous genotype and T:T homozygous genotype, verified by resequencing.

[0042] Forty maize inbred lines from Example 1 were selected and KASP marker typing verification was performed using the above-mentioned kit. The specific operation steps are as follows: Use genomic DNA from each sample as a template to prepare a 10 μL PCR amplification reaction system. The reaction system contains 2 μL of genomic DNA at a concentration of 4 to 50 ng / μL, 0.14 μL of primer mixture, 5 μL of ProbeMixA solution at a 2-fold concentration, and 3 μL of enzyme-free water. Place the reaction system in a real-time PCR instrument for amplification.

[0043] The PCR amplification reaction program was set as follows: Pre-denaturation at 95°C for 10 min. Then, the first-stage amplification program was implemented: denaturation at 95°C for 20 s, annealing at 61°C for 40 s, for a total of 10 cycles, with the annealing temperature decreasing by 0.6°C per cycle. Following this, the second-stage amplification program was implemented: denaturation at 95°C for 20 s, annealing at 55°C for 40 s, for a total of 31 cycles. After amplification, the product was incubated at 25°C for 10 min and then stored at 4°C in the dark.

[0044] After the amplification reaction, the PCR plate was placed in an ABI 7500 qPCR instrument and incubated at 35°C for 30 seconds before fluorescence signals were acquired. FAM fluorescence signals were detected at excitation wavelengths of 485 nm and emission wavelengths of 520 nm. HEX fluorescence signals were detected at excitation wavelengths of 528 nm and emission wavelengths of 560 nm. The HEX and FAM fluorescence values ​​for each well, as well as the fluorescence value of the reference dye ROX, were derived. The HEX and FAM fluorescence values ​​were standardized based on the ROX fluorescence value to obtain the relative fluorescence value for each well. Cluster analysis was performed on the samples based on the relative fluorescence values.

[0045] Test results as follows Figure 6 As shown, by Figure 6 It can be seen that this locus has two genotypes: blue dots represent C:C and red dots represent T:T. This is divided into two haps: C:C corresponds to hap1, and T:T corresponds to hap2, and then correlated with the phenotypes of the two locations, such as... Figure 7 As shown, the genotyping results indicate that all samples can be clearly divided into two clusters. The blue clusters closer to the X-axis correspond to FAM fluorescence signals, representing C:C homozygous genotypes, corresponding to haplotype Hap1. The red clusters closer to the Y-axis correspond to HEX fluorescence signals, representing T:T homozygous genotypes, corresponding to haplotype Hap2. The black dots represent negative controls, located near the origin of the coordinate system.

[0046] Comparison of KASP genotyping results with resequencing genotypes showed an overall concordance rate of over 98%, with only a very small number of inconsistent samples due to DNA quality issues, indicating that the marker has high genotyping accuracy. Association analysis between the genotyping results and the plant height phenotypes of the two pilot projects revealed that the average plant height of the C:C homozygous genotype material was significantly higher than that of the T:T homozygous genotype material, with both differences reaching a highly significant level. This demonstrates that the marker can be accurately used for genotyping and early-stage assisted selection of maize plant height.

[0047] The results of KASP molecular marker scanning for maize plant height are shown in the table below. In the table, / and not detected may not have been detected due to DNA quality issues; · indicates that resequencing data for that site is missing.

[0048] The SNP locus provided by this invention is directly located in the coding region of the maize gene Zm00001eb259660. It is a functional missense mutation that can lead to changes in the amino acid sequence of the encoded protein, rather than a genomic spacer variant indirectly linked to the trait. This eliminates the risk of segregation between the marker and the target trait due to genetic recombination from a genetic perspective. Compared to existing plant height-related markers obtained solely based on statistical association, the marker locus of this invention has zero genetic distance from the causative variation controlling plant height. When applied in breeding populations with different genetic backgrounds, there will be no decoupling between the marker and the trait, significantly improving selection accuracy. Furthermore, this locus has been validated through association between multiple field phenotypes and natural populations, showing a stable genetic effect on plant height under different planting environments. There are no effect fluctuations caused by environmental interactions, and it can stably distinguish phenotypic differences between different alleles, solving the technical problems of poor universality and unstable genetic effects of existing plant height markers.

[0049] This invention utilizes a KASP molecular marker developed based on a target SNP locus. Primers are designed to target conserved genomic sequences flanking the locus, resulting in high amplification specificity and clear, definitive genotyping results. It can reliably distinguish between two homozygous alleles, with genotyping results showing over 98% consistency with whole-genome resequencing, demonstrating high detection reliability. Detection of this marker can be completed using only a conventional quantitative real-time PCR platform, eliminating the need for costly gene chips or high-throughput sequencing platforms. The single-sample detection cycle is short and cost-effective, with a standardized procedure that eliminates the need for subsequent gel electrophoresis or sequencing steps. This makes it suitable for batch detection of large-scale segregating populations in breeding processes, solving the technical problems of high cost, complex procedures, and difficulty in applying existing plant height-related marker detection methods to field breeding practices.

[0050] The molecular marker provided by this invention enables early genotypic selection of maize plant height, allowing genotypic identification of target loci during the seedling stage without waiting for physiological maturity for plant height phenotypic surveys, effectively shortening the breeding selection cycle. Simultaneously, this marker can be directly used for targeted molecular improvement of maize plant height. Through marker-assisted backcrossing, the T allele controlling lower plant height can be precisely introduced, reducing the workload of phenotypic identification during backcrossing, minimizing the interference of environmental factors on selection results, and improving the accuracy and efficiency of plant height trait improvement. Furthermore, this marker can be used for large-scale genotypic surveys of maize germplasm resources to rapidly identify germplasm materials carrying superior alleles, providing new technical support and material basis for breeding ideal maize plant types.

[0051] 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. A KASP molecular marker PH5-KASP based on the SNP site of the maize Zm00001eb259660 gene, characterized in that, The KASP molecular marker specifically targets a single nucleotide polymorphism (SNP) site at 226,029,364 bp on chromosome 5 of maize. The SNP site is located in the coding region of exon 1 of the T001 transcript of the maize Zm00001eb259660 gene and is a missense mutation of c.485C>T. The KASP molecular marker consists of a first allele-specific forward primer, a second allele-specific forward primer, and a universal reverse primer. The first allele-specific forward primer carries a FAM fluorescent tag sequence and specifically binds to the upstream flanking sequence of the C allele at the SNP site. The second allele-specific forward primer carries a HEX fluorescent tag sequence and specifically binds to the upstream flanking sequence of the T allele at the SNP site. The universal reverse primer binds to the conserved genomic sequence downstream of the SNP site.

2. The KASP molecular marker PH5-KASP based on the SNP site of the maize Zm00001eb259660 gene according to claim 1, characterized in that, The nucleotide sequence of the first allele-specific forward primer is 5'-GAAGGTGACCAAGTTCATGCTTACATCGAATGCAGTTCAAAAAC-3', the nucleotide sequence of the second allele-specific forward primer is 5'-GAAGGTCGGAGTCAACGGATTTACATCGAATGCAGTTCAAAAAT-3', and the nucleotide sequence of the universal reverse primer is 5'-GCTGGGGCGACAGTACCT-3'.

3. The KASP molecular marker PH5-KASP based on the SNP site of the maize Zm00001eb259660 gene according to claim 1, characterized in that, The SNP locus was derived from the association analysis results of multi-point phenotypes and whole-genome resequencing of 377 maize inbred lines. It is located within a linkage disequilibrium range of 400kb upstream and downstream of the significant association interval for plant height, and is the only functional variant locus in the coding region of candidate gene Zm00001eb259660 that is highly significantly associated with plant height.

4. The KASP molecular marker PH5-KASP based on the SNP site of the maize Zm00001eb259660 gene according to claim 1, characterized in that, The c.485C>T missense mutation causes the 162nd amino acid residue of the protein encoded by the maize Zm00001eb259660 gene to be replaced by isoleucine.

5. A kit for identifying maize plant height genotypes based on the KASP molecular marker PH5-KASP at the SNP site of the maize Zm00001eb259660 gene, as described in any one of claims 1 to 4, characterized in that, The kit also includes PCR amplification reagents, fluorescence signal detection reagents, negative controls and positive controls; the PCR amplification reagents contain 2×ProbeMixA solution and enzyme-free water ddH2O, and the KASP molecular marker PH5-KASP is aliquoted and stored in the form of premixed primer mix.

6. The maize plant height genotype identification kit according to claim 5, characterized in that, The premixed primer Mix was prepared by mixing 6 μl of first allele-specific forward primer, 6 μl of second allele-specific forward primer, 15 μl of universal reverse primer, and 23 μl of enzyme-free water (ddH2O) in a volume ratio; the negative control was template-free enzyme-free water (ddH2O), and the positive control was maize genomic DNA of C:C homozygous genotype and T:T homozygous genotype verified by resequencing.

7. The application of the KASP molecular marker PH5-KASP as described in any one of claims 1 to 4 or the maize plant height genotype identification kit as described in any one of claims 5 to 6 in the detection of maize plant height-related genotypes, the general survey of maize germplasm resource plant height trait genotypes, and maize molecular marker-assisted breeding.

8. The application according to claim 7, characterized in that, The application includes detecting the genotype of the maize material to be tested at the 226,029,364bp site on chromosome 5 of maize, and classifying the maize material to be tested into two haplotypes based on the detection results, wherein the C:C homozygous genotype corresponds to haplotype Hap1, and the T:T homozygous genotype corresponds to haplotype Hap2.

9. The application according to claim 7, characterized in that, The method for detecting the genotype of maize material to be tested includes the following steps: S1: Genomic DNA was extracted from fresh leaves of maize seedlings using a modified CTAB method. After quality testing by agarose gel electrophoresis and UV spectrophotometer, the DNA concentration was uniformly adjusted to 4-50 ng / μl. S2: Using the genomic DNA extracted in step S1 as a template, PCR amplification reaction is performed using the KASP molecular marker PH5-KASP; S3: After the PCR amplification reaction was completed, the amplification products were incubated at 35℃ for 30s, and the FAM fluorescence signal and HEX fluorescence signal were collected respectively. The FAM fluorescence signal was detected at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescence signal was detected at an excitation wavelength of 528nm and an emission wavelength of 560nm. S4: The collected signals were standardized based on the fluorescence value of the reference dye ROX, and the genotype of the maize material to be tested was determined by cluster analysis.

10. The application according to claim 9, characterized in that, The PCR amplification reaction system in step S2, in a total volume of 10 μl, consists of: 2 μl of genomic DNA at a concentration of 4-50 ng / μl, 0.14 μl of premixed primers Mix, 5 μl of 2×ProbeMixA solution, and 3 μl of enzyme-free water ddH2O. The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s, 61℃ annealing for 40 s, for a total of 10 cycles; 95℃ denaturation for 20 s, 55℃ annealing for 40 s, for a total of 31 cycles; incubation at 25℃ for 10 min; and storage of the amplification products at 4℃ in the dark.

Images