A SNP molecular marker combination related to corn flowering period, a gene chip and application thereof

Abstract

This invention provides a combination of SNP molecular markers related to maize flowering time, a gene chip, and their applications, relating to the field of plant molecular breeding technology. It includes 58 SNP molecular markers, as shown in SNP1 to SNP58. These molecular markers efficiently and accurately identify maize flowering time-related genotypes. The developed flowering time haplotype molecular markers reveal the genetic variation characteristics of maize flowering time, significantly improving the accuracy of identifying minor gene functional sites. This can be used for further development of functional sites in maize microarrays, laying the foundation for breeding varieties with multiple superior loci aggregation, and providing a novel tool for marker-assisted selection breeding. It solves the technical problem that existing single-marker detection systems based on a few markers often produce false negative results due to primer failure or allele loss, making it difficult to comprehensively and accurately reflect the variation of genes related to flowering time.

Description

Technical Field

[0001] This invention relates to the field of plant molecular breeding technology, and in particular to a combination of SNP molecular markers related to maize flowering period, a gene chip, and their applications. Background Technology

[0002] Maize is an important food crop in my country, widely cultivated, and its phenotypic traits are subject to diverse requirements from different ecological environments. Flowering time is a key agronomical trait of maize, directly affecting its ecological adaptability. On the one hand, when faced with abiotic stresses such as drought and high temperatures, plants can adjust their flowering time to avoid unfavorable environments and ensure the successful completion of reproductive growth. On the other hand, maize is a typical short-day crop, extremely sensitive to photoperiod; it can only flower and bear fruit normally when the day length is shorter than the critical day length. Extending the day duration inhibits flowering, while shortening it promotes it. Current research shows that many cloned flowering time genes are involved in regulating maize's photoperiod sensitivity, thus affecting its environmental adaptability. Systematically mining flowering time-related genes and using genotyping technology to directionally improve flowering time at the genetic level is of great significance for accelerating the creation of superior germplasm resources and promoting the cross-ecological dissemination and promotion of superior varieties.

[0003] Genome-wide association analysis (GWAS) is a highly efficient method for elucidating the genetic basis of complex traits. Its core principle is to utilize high-throughput genotyping technology to detect tens of thousands of single nucleotide polymorphisms (SNPs) in natural populations, and then combine this with phenotypic data of the target trait to employ statistical models to uncover population-level associations between genotypes and phenotypes. Compared to traditional linkage analysis, this method does not require the construction of segregating populations, can directly utilize maize inbred lines, and can simultaneously analyze the genetic effects controlled by multiple minor genes, providing a foundation for the subsequent development of molecular markers related to flowering time.

[0004] Currently, screening suitable flowering materials for different ecological environments still faces many technical challenges. Traditional phenotypic evaluation methods are easily affected by environmental interference, and are time-consuming and labor-intensive. Meanwhile, single-molecule marker detection systems based on a few markers are prone to false negatives due to primer failure or allele loss, making it difficult to comprehensively and accurately reflect the variation of genes related to flowering. These shortcomings limit the accurate capture and breeding application of small-scale multi-gene loci.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] One of the objectives of this invention is to provide a combination of SNP molecular markers related to the flowering period of maize, in order to solve the technical problem that existing single-molecule marker detection systems based on a few markers are prone to false negative results due to primer failure or allele loss, making it difficult to comprehensively and accurately reflect the variation of genes related to the flowering period.

[0007] The second objective of this invention is to provide a gene chip related to the flowering period of maize.

[0008] The third objective of this invention is to provide a reagent kit.

[0009] The fourth objective of this invention is to provide the application of any one of the above-mentioned SNP molecular marker combinations, the above-mentioned gene chip, and the above-mentioned kit in genome-wide association analysis during the flowering period of maize.

[0010] The fifth objective of this invention is to provide the application of any one of the above-mentioned SNP molecular marker combination, the above-mentioned gene chip, and the above-mentioned kit in identifying the flowering period of maize.

[0011] The sixth objective of this invention is to provide a method for identifying or assisting in the identification of the flowering period of corn.

[0012] The seventh objective of this invention is to provide the application of the above-mentioned method in maize breeding.

[0013] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a combination of SNP molecular markers related to the flowering period of maize, the combination of SNP molecular markers including 58 SNP molecular markers, the 58 SNP molecular markers being shown as SNP1 to SNP58; The physical location information of SNP1~SNP58 is as follows: The genomic location of SNP1 is Chr4: 166252736 bp; The genomic location of SNP2 is Chr4: 166253066 bp; The genomic location of SNP3 is Chr4: 166253772 bp; The genomic location of SNP4 is Chr4: 166255095 bp; The genomic location of SNP5 is Chr4: 166255271 bp; The genomic location of SNP6 is Chr4: 166255526 bp; The genomic location of SNP7 is Chr4: 166255612 bp; The genomic location of SNP8 is Chr4: 166255693 bp; The genomic location of SNP9 is Chr4: 166256979 bp; The genomic location of SNP10 is Chr4: 166257205 bp; The genomic location of SNP11 is Chr4: 166257601 bp; The genomic location of SNP12 is Chr4: 166257607 bp; The genomic location of SNP13 is Chr4: 166258207 bp; The genomic location of SNP14 is Chr4: 166258317 bp; The genomic location of SNP15 is Chr4: 166258530 bp; The genomic location of SNP16 is Chr4: 166258647 bp; The genomic location of SNP17 is Chr4: 166258770 bp; The genomic location of SNP18 is Chr4: 166259704 bp; The genomic location of SNP19 is Chr4: 166260070 bp; The genomic location of SNP20 is Chr4: 166260170 bp; The genomic location of SNP21 is Chr4: 166261854 bp; The genomic location of SNP22 is Chr4: 166262627 bp; The genomic location of SNP23 is Chr4: 166262641 bp; The genomic location of SNP24 is Chr4: 166262842 bp; The genomic location of SNP25 is Chr4: 166263015 bp; The genomic location of SNP26 is Chr4: 166263055 bp; The genomic location of SNP27 is Chr4: 166263307 bp; The genomic location of SNP28 is Chr4: 166263643 bp; The genomic location of SNP29 is Chr4: 166263960 bp; The genomic location of SNP30 is Chr4: 166264069 bp; The genomic location of SNP31 is Chr4: 166264127 bp; The genomic location of SNP32 is Chr4: 166264156 bp; The genomic location of SNP33 is Chr4: 166264162 bp; The genomic location of SNP34 is Chr4: 166264176 bp; The genomic location of SNP35 is Chr4: 166264306 bp; The genomic location of SNP36 is Chr4: 166264316 bp; The genomic location of SNP37 is Chr4: 166264357 bp; The genomic location of SNP38 is Chr4: 166264406 bp; The genomic location of SNP39 is Chr4: 166264462 bp; The genomic location of SNP40 is Chr4: 166264566 bp; The genomic location of SNP41 is Chr4: 166264882 bp; The genomic location of SNP42 is Chr4: 166265112 bp; The genomic location of SNP43 is Chr4: 166265192 bp; The genomic location of SNP44 is Chr4: 166278814 bp; The genomic location of SNP45 is Chr4: 166281349 bp; The genomic location of SNP46 is Chr4: 166281456 bp; The genomic location of SNP47 is Chr4: 166281473 bp; The genomic location of SNP48 is Chr4: 166281528 bp; The genomic location of SNP49 is Chr4: 166282065 bp; The genomic location of SNP50 is Chr4: 166282442 bp; The genomic location of SNP51 is Chr4: 166282487 bp; The genomic location of SNP52 is Chr4: 166282881 bp; The genomic location of SNP53 is Chr4: 166283036 bp; The genomic location of SNP54 is Chr4: 166283267 bp; The genomic location of SNP55 is Chr4: 166283416 bp; The genomic location of SNP56 is Chr4: 166283641 bp; The genomic location of SNP57 is Chr4: 166283653 bp; The genomic location of SNP58 is Chr4: 166283818 bp; The physical location information of the 58 SNP molecular markers was determined based on the maize B73 RefGen_v4 genome.

[0014] Secondly, the present invention provides a gene chip related to the flowering period of maize, including probes and / or primers for detecting the above-mentioned SNP molecular marker combinations.

[0015] Furthermore, the gene chip is a solid-phase chip.

[0016] Thirdly, the present invention provides a kit comprising probes and / or primers for detecting the above-described SNP molecular marker combinations.

[0017] Fourthly, the present invention provides the application of any one of the above-mentioned SNP molecular marker combinations, the above-mentioned gene chip, and the above-mentioned kit in genome-wide association analysis during the flowering period of maize.

[0018] Fifthly, the present invention provides the application of any one of the above-mentioned SNP molecular marker combination, the above-mentioned gene chip, and the above-mentioned kit in identifying the flowering period of maize.

[0019] In a sixth aspect, the present invention provides a method for identifying or assisting in the identification of the flowering period of maize, comprising detecting the genotype of the above-mentioned SNP molecular marker combination in a maize sample to be tested, and identifying or assisting in the identification of the flowering period of maize based on the genotype of the maize sample to be tested. When the haplotype sequence formed by SNP1 to SNP58 is as shown in SEQ ID NO.1, the flowering period of maize is early flowering phenotype.

[0020] Furthermore, the corn in question is a maize inbred line.

[0021] In a seventh aspect, the present invention provides the application of the above-described method in maize breeding.

[0022] Furthermore, the maize breeding includes selection based on maize flowering time.

[0023] This invention provides a combination of SNP molecular markers related to maize flowering time. Using inbred lines of major domestic varieties as research materials, the relevant genes / locusts can be directly used for variety screening and improvement. Through genome-wide association analysis, it can better uncover minor genes and analyze the relationships between multiple genetic loci than linkage mapping. Molecular markers are used to efficiently and accurately identify maize flowering time-related genotypes. The developed flowering time haplotype molecular markers reveal the genetic variation characteristics of maize flowering time, significantly improving the accuracy of identifying minor gene functional loci. This can be used for further development of maize microarray functional loci, laying the foundation for breeding varieties with multiple superior loci aggregation, and providing a new tool for marker-assisted selection breeding. This invention solves the technical problem that existing single-marker detection systems based on a few markers often produce false negative results due to primer failure or allele loss, making it difficult to comprehensively and accurately reflect the variation of flowering time-related genes. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 This is a genome-wide association analysis diagram of the Xinxiang sample provided in Embodiment 1 of the present invention, wherein A is a Manhattan diagram and B is a QQ diagram; Figure 2 The interval distribution diagram of Zm00001d051670, Zm00001d051671, Zm00001d051672, Zm00001d051673 and Zm00001d051674 provided in Embodiment 1 of the present invention; Figure 3 The expression spectra of Zm00001d051670, Zm00001d051671, Zm00001d051672, Zm00001d051673 and Zm00001d051674 provided in Embodiment 1 of the present invention; Figure 4 The figures show the genotypes and flowering time differences of the two haplotypes provided in Embodiment 2 of the present invention, wherein A represents the genotypes and frequencies of the two haplotypes, and B represents the phenotypic differences of the two haplotypes. Figure 5 This is a genome-wide association analysis diagram of the Fengcheng sample provided in Embodiment 3 of the present invention, wherein A is a Manhattan diagram and B is a QQ diagram; Figure 6The two haplotype phenotypic differences of the Fengcheng sample provided in Embodiment 3 of the present invention. Detailed Implementation

[0026] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0027] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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.

[0029] This invention conducts genome-wide association analysis based on SNP locus information and flowering period phenotypic data from 484 important maize parent lines, obtaining SNP markers significantly associated with maize flowering period, and obtaining haplotype molecular markers closely linked to maize flowering period associated loci through linkage disequilibrium analysis. This set of haplotype molecular markers can be used for maize flowering period identification and improvement.

[0030] In one aspect, the present invention provides a combination of SNP molecular markers related to the flowering period of maize, the combination of SNP molecular markers including 58 SNP molecular markers, as shown in SNP1 to SNP58; The physical location information of SNP1~SNP58 is as follows: The genomic location of SNP1 is Chr4: 166252736 bp; The genomic location of SNP2 is Chr4: 166253066 bp; The genomic location of SNP3 is Chr4: 166253772 bp; The genomic location of SNP4 is Chr4: 166255095 bp; The genomic location of SNP5 is Chr4: 166255271 bp; The genomic location of SNP6 is Chr4: 166255526 bp; The genomic location of SNP7 is Chr4: 166255612 bp; The genomic location of SNP8 is Chr4: 166255693 bp; The genomic location of SNP9 is Chr4: 166256979 bp; The genomic location of SNP10 is Chr4: 166257205 bp; The genomic location of SNP11 is Chr4: 166257601 bp; The genomic location of SNP12 is Chr4: 166257607 bp; The genomic location of SNP13 is Chr4: 166258207 bp; The genomic location of SNP14 is Chr4: 166258317 bp; The genomic location of SNP15 is Chr4: 166258530 bp; The genomic location of SNP16 is Chr4: 166258647 bp; The genomic location of SNP17 is Chr4: 166258770 bp; The genomic location of SNP18 is Chr4: 166259704 bp; The genomic location of SNP19 is Chr4: 166260070 bp; The genomic location of SNP20 is Chr4: 166260170 bp; The genomic location of SNP21 is Chr4: 166261854 bp; The genomic location of SNP22 is Chr4: 166262627 bp; The genomic location of SNP23 is Chr4: 166262641 bp; The genomic location of SNP24 is Chr4: 166262842 bp; The genomic location of SNP25 is Chr4: 166263015 bp; The genomic location of SNP26 is Chr4: 166263055 bp; The genomic location of SNP27 is Chr4: 166263307 bp; The genomic location of SNP28 is Chr4: 166263643 bp; The genomic location of SNP29 is Chr4: 166263960 bp; The genomic location of SNP30 is Chr4: 166264069 bp; The genomic location of SNP31 is Chr4: 166264127 bp; The genomic location of SNP32 is Chr4: 166264156 bp; The genomic location of SNP33 is Chr4: 166264162 bp; The genomic location of SNP34 is Chr4: 166264176 bp; The genomic location of SNP35 is Chr4: 166264306 bp; The genomic location of SNP36 is Chr4: 166264316 bp; The genomic location of SNP37 is Chr4: 166264357 bp; The genomic location of SNP38 is Chr4: 166264406 bp; The genomic location of SNP39 is Chr4: 166264462 bp; The genomic location of SNP40 is Chr4: 166264566 bp; The genomic location of SNP41 is Chr4: 166264882 bp; The genomic location of SNP42 is Chr4: 166265112 bp; The genomic location of SNP43 is Chr4: 166265192 bp; The genomic location of SNP44 is Chr4: 166278814 bp; The genomic location of SNP45 is Chr4: 166281349 bp; The genomic location of SNP46 is Chr4: 166281456 bp; The genomic location of SNP47 is Chr4: 166281473 bp; The genomic location of SNP48 is Chr4: 166281528 bp; The genomic location of SNP49 is Chr4: 166282065 bp; The genomic location of SNP50 is Chr4: 166282442 bp; The genomic location of SNP51 is Chr4: 166282487 bp; The genomic location of SNP52 is Chr4: 166282881 bp; The genomic location of SNP53 is Chr4: 166283036 bp; The genomic location of SNP54 is Chr4: 166283267 bp; The genomic location of SNP55 is Chr4: 166283416 bp; The genomic location of SNP56 is Chr4: 166283641 bp; The genomic location of SNP57 is Chr4: 166283653 bp; The genomic location of SNP58 is Chr4: 166283818 bp; The physical location information of the 58 SNP molecular markers was determined based on the maize B73 RefGen_v4 genome.

[0031] This invention uses inbred lines of major domestic varieties as research materials, and the relevant genes / locuses can be directly used for variety screening and improvement. Utilizing genome-wide association analysis (GWAS), it can better uncover minor genes and analyze the relationships between multiple genetic loci than linkage mapping. Molecular markers are used to efficiently and accurately identify genotypes related to maize flowering time. The developed flowering time haplotype molecular markers reveal the genetic variation characteristics of maize flowering time, significantly improving the accuracy of identifying minor gene functional loci. This can be used for further development of maize microarray functional loci, laying the foundation for breeding varieties with multiple superior loci aggregation, and providing a new tool for marker-assisted selection breeding. This invention solves the technical problem that existing single-marker detection systems based on a few markers often produce false negative results due to primer failure or allele loss, making it difficult to comprehensively and accurately reflect the variation of genes related to flowering time.

[0032] According to another aspect of the present invention, a gene chip related to the flowering period of maize is also provided, including probes and / or primers for detecting the above-mentioned SNP molecular marker combinations.

[0033] In some specific implementations, the gene chip is a solid-phase chip.

[0034] According to another aspect of the present invention, a kit is also provided, comprising probes and / or primers for detecting the above-described SNP molecular marker combinations.

[0035] According to another aspect of the present invention, the application of any one of the above-described SNP molecular marker combinations, the above-described gene chip, and the above-described kit in genome-wide association analysis during maize flowering is also provided.

[0036] According to another aspect of the present invention, the application of any one of the above-described SNP molecular marker combination, the above-described gene chip, and the above-described kit in identifying the flowering period of maize is also provided.

[0037] According to another aspect of the present invention, a method for identifying or assisting in the identification of maize flowering period is also provided, comprising detecting the genotype of the above-mentioned SNP molecular marker combination in a maize sample to be tested, and identifying or assisting in the identification of maize flowering period based on the genotype of the maize sample to be tested. When the haplotype sequence formed by SNP1 to SNP58 is as shown in SEQ ID NO.1, the flowering period of maize is the early flowering phenotype.

[0038] In some specific implementations, the corn is a maize inbred line.

[0039] According to another aspect of the present invention, the application of the above-described method in maize breeding is also provided.

[0040] In some specific implementations, the maize breeding includes selection based on the maize flowering period.

[0041] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0042] In the following examples, the 484 maize inbred lines are maize B73, maize W22, maize B104, maize Chang 7-2, maize Dan 340, maize Qi 319, maize Ye 478 and maize Zheng 58.

[0043] Example 1: Genome-wide association analysis of maize flowering period and analysis of the Chr4_166253066 locus. 1. Phenotypic Acquisition In 2024, 484 core inbred lines from various maize-producing regions across China were planted in Xinxiang, Henan Province, during the peak maize season in the Huang-Huai-Hai Plain. The experiment employed a completely randomized block design with three biological replicates. Each material was planted in one row, 3.5 meters long, with 15 plants per row, resulting in a planting density of 4500 plants per mu (approximately 667 hectares). Flowering was defined as when more than half of the plants in the tassel had shed pollen. Observations were made daily, and the date when more than 50% of the plants in a row reached this standard was recorded as the flowering date for that material. The phenotypic values ​​of flowering date from the three biological replicates for each inbred line were then corrected. First, the deviation of each replicate from the median was calculated. Individual abnormal replicates with significant deviations (more than twice the standard deviation) were removed. The arithmetic mean of the remaining replicates was then used as the final phenotypic value for that material for association analysis.

[0044] 2. Genotyping Fresh leaves from the 484 inbred lines were collected, and genomic DNA was extracted using the alkaline lysis method. Genotyping was performed using a Maize 50K high-density SNP microarray (containing 50K marker sites) strictly following the Illumina Infinium microarray detection standard procedure, ultimately obtaining 36,896 high-quality SNP markers covering the entire genome. PLINK software was used for quality control of the genotype data, removing SNP sites and samples with a deletion rate greater than 10%. Subsequently, Beagle software was used to fill in the remaining missing genotypes, ultimately obtaining 35,986 high-quality SNP markers from the 484 inbred lines.

[0045] 3. Correlation Analysis Using 35,986 SNPs as genotypic data and the mean flowering time after treatment as the phenotype, genome-wide association analysis was performed using GAPIT software with various models including General Linear Model (GLM), Mixed Linear Model (MLM), Multiple Locus Mixture Model (MLMM), FarmCPU (Fixed and random model Circulating Probability Unification), and BLINK (Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway). The effectiveness of false positive and false negative control was evaluated by comparing the Quantile-Quantile (QQ) plots of each model: In the ideal model, when -log10(P) of the observed and expected values ​​is less than 3, the scatter plots should closely follow the diagonal, indicating good control of population stratification and kinship, and low false positives; while when -log10(P) is greater than 3, the scatter plots begin to tilt upwards and deviate significantly from the diagonal, reflecting that the model can sensitively detect true association signals, and has better false negative control. After comprehensive comparison, the QQ plot of the BLINK model shows that the low-value area is close to the diagonal and the high-value area is significantly upturned, achieving the best balance between false positives and false negatives. Therefore, the BLINK model was selected for the final association analysis.

[0046] Based on the BLINK model analysis, a total of 19 SNP sites significantly associated with flowering period were detected (using Bonferroni correction threshold P=2.47×10⁻⁶). -4 )(like Figure 1 As shown in the figure, a SNP locus highly significantly associated with maize flowering time was detected at position 166253066 bp on chromosome 4 (P=8.94×10). -5 ).

[0047] Based on the reference genome sequence of maize inbred line B73, gene annotation searches were performed within a 100 kb region upstream and downstream of this SNP site. The results showed that only 5 annotated genes existed in this region: Zm00001d051670, Zm00001d051671, Zm00001d051672, Zm00001d051673, and Zm00001d051674 (e.g., Zm00001d051670, Zm00001d051671, Zm00001d051672, Zm00001d051673, and Zm00001d051674). Figure 2(As shown). Among them, Zm00001d051673 encodes tRNA, which belongs to the redundant basic translational elements in the cell and can be preferentially excluded. According to the published RNA-seq expression data, Zm00001d051670 is expressed in pollen; Zm00001d051671 is constitutively expressed and is expressed in all parts; Zm00001d051672 is mainly expressed in roots, female spikelets and filaments; Zm00001d051674 is mainly expressed in spike primordia and grains (e.g., Figure 3 As shown in the figure, the horizontal axis 1-21 corresponds to numbers 1-21 in Table 1. Meanwhile, gene function annotation shows that Zm00001d051670 encodes a plant immune regulatory hub protein (RPM1-interacting protein 4); Zm00001d051671 encodes the core subunit of the APC / C complex, which is crucial for cell cycle and developmental transition; direct functional validation evidence in tomato and Arabidopsis supports APC / C regulation of flowering period; Zm00001d051672 encodes a GPI-anchoring protein (LORELEI-like), which assists in pollen tube reception and fertilization; Zm00001d051674 encodes a phosphatidylinositol / phosphatidylcholine transporter (Sec14 family), which functions in lipid molecule transport. Based on the above expression specificity and functional annotation information, it is speculated that Zm00001d051671 is a key functional gene regulating flowering period at this site.

[0048] Table 1

[0049] Example 2: Development of haplotype markers based on flowering-related loci 1. Download genotype data of associated populations containing 540 maize inbred lines from the Maizego database (http: / / www.maizego.org / ), and extract all SNPs within a 1 Mb interval upstream and downstream of the chr4_166253066 locus on chromosome 4, obtaining a total of 4705 SNP loci (including chr4_166253066 itself).

[0050] 2. After basic quality control of the above 4705 SNP loci, linkage disequilibrium analysis was performed using TASSEL 5 software. The coefficient of determination (CDR) for linkage disequilibrium between the Chr4_166253066 locus and other loci was calculated, and the r values ​​were selected. 2Linked SNPs with a value greater than 0.8 (values ​​between 0 and 1 indicate the degree of linkage between two SNPs, and a value equal to 1 indicates that the two SNPs are completely linked) were identified. A total of 58 SNPs closely linked to Chr4_166253066 (including Chr4_166253066 itself) were identified, as shown in Table 2.

[0051] Table 2

[0052] 3. Genotypes of the above 58 SNP loci (including Chr4_166253066) were extracted from 540 inbred lines. Haplotype analysis was performed using Haploview software to obtain two major haplotypes, Hap1 and Hap2 (e.g., Figure 4 (A). Through examining the flowering phenotypes of corresponding materials in the associated populations, it was found that individuals with the Hap1 genotype generally exhibited early flowering, while individuals with the Hap2 genotype generally exhibited late flowering, and the difference in flowering time between the two groups reached a highly significant level (P<0.001). Figure 4 (B). Therefore, when the haplotype of the above SNP marker is the Hap1 genotype, it is maize early-flowering phenotype germplasm. The physical location of the SNP locus is determined based on the maize B73RefGen_v4 genome. The multi-locus haplotype markers developed based on this locus and its linked loci can effectively distinguish between early-flowering and late-flowering maize germplasm and have clear breeding application value.

[0053] Example 3: Multi-site verification of flowering-related loci and their haplotype markers 1. In 2024, 484 identical inbred lines were planted in Fengcheng, Liaoning Province, using the same experimental design. A survey of the flowering period (pollen shedding period) was conducted, and GWAS and related analyses were performed. The results showed that, using the same BLINK model analysis, the same significant signal was found at position 166253066 on chromosome 4 (P=1.33×10⁻⁶). -4 )(like Figure 5 (As shown).

[0054] 2. Based on the haplotype molecular marker Hap1, 484 inbred lines were divided into two groups for t-test. The haplotype grouping results are as follows: Figure 6 As shown, the flowering time of the two inbred lines differed significantly (p = 2.7 × 10⁻⁶). -4 This further illustrates the association between the haplotype marker and the flowering period. Genotyping of the haplotype marker can be used to determine and improve the flowering period of inbred lines.

[0055] Hap1: GGCGCGGCCAGGCGCGGCATGAGGGGGCCCCCCCGACGGGCGACCGGGGGGTCCACCT (SEQ ID NO. 1).

[0056] Hap2: CATATAATTGATTATAATGAAGAATAAAGTTATTAGTAATTAGTTCAAATTGATTTTC (SEQ ID NO. 2).

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A reagent for detecting SNP molecular marker combinations associated with maize flowering period in the identification or auxiliary identification of maize flowering period, characterized in that, The SNP molecular marker combination includes 58 SNP molecular markers, as shown in SNP1 to SNP58. The physical location information of SNP1~SNP58 is as follows: The genomic location of SNP1 is Chr4: 166252736 bp; The genomic location of SNP2 is Chr4: 166253066 bp; The genomic location of SNP3 is Chr4: 166253772 bp; The genomic location of SNP4 is Chr4: 166255095 bp; The genomic location of SNP5 is Chr4: 166255271 bp; The genomic location of SNP6 is Chr4: 166255526 bp; The genomic location of SNP7 is Chr4: 166255612 bp; The genomic location of SNP8 is Chr4: 166255693 bp; The genomic location of SNP9 is Chr4: 166256979 bp; The genomic location of SNP10 is Chr4: 166257205 bp; The genomic location of SNP11 is Chr4: 166257601 bp; The genomic location of SNP12 is Chr4: 166257607 bp; The genomic location of SNP13 is Chr4: 166258207 bp; The genomic location of SNP14 is Chr4: 166258317 bp; The genomic location of SNP15 is Chr4: 166258530 bp; The genomic location of SNP16 is Chr4: 166258647 bp; The genomic location of SNP17 is Chr4: 166258770 bp; The genomic location of SNP18 is Chr4: 166259704 bp; The genomic location of SNP19 is Chr4: 166260070 bp; The genomic location of SNP20 is Chr4: 166260170 bp; The genomic location of SNP21 is Chr4: 166261854 bp; The genomic location of SNP22 is Chr4: 166262627 bp; The genomic location of SNP23 is Chr4: 166262641 bp; The genomic location of SNP24 is Chr4: 166262842 bp; The genomic location of SNP25 is Chr4: 166263015 bp; The genomic location of SNP26 is Chr4: 166263055 bp; The genomic location of SNP27 is Chr4: 166263307 bp; The genomic location of SNP28 is Chr4: 166263643 bp; The genomic location of SNP29 is Chr4: 166263960 bp; The genomic location of SNP30 is Chr4: 166264069 bp; The genomic location of SNP31 is Chr4: 166264127 bp; The genomic location of SNP32 is Chr4: 166264156 bp; The genomic location of SNP33 is Chr4: 166264162 bp; The genomic location of SNP34 is Chr4: 166264176 bp; The genomic location of SNP35 is Chr4: 166264306 bp; The genomic location of SNP36 is Chr4: 166264316 bp; The genomic location of SNP37 is Chr4: 166264357 bp; The genomic location of SNP38 is Chr4: 166264406 bp; The genomic location of SNP39 is Chr4: 166264462 bp; The genomic location of SNP40 is Chr4: 166264566 bp; The genomic location of SNP41 is Chr4: 166264882 bp; The genomic location of SNP42 is Chr4: 166265112 bp; The genomic location of SNP43 is Chr4: 166265192 bp; The genomic location of SNP44 is Chr4: 166278814 bp; The genomic location of SNP45 is Chr4: 166281349 bp; The genomic location of SNP46 is Chr4: 166281456 bp; The genomic location of SNP47 is Chr4: 166281473 bp; The genomic location of SNP48 is Chr4: 166281528 bp; The genomic location of SNP49 is Chr4: 166282065 bp; The genomic location of SNP50 is Chr4: 166282442 bp; The genomic location of SNP51 is Chr4: 166282487 bp; The genomic location of SNP52 is Chr4: 166282881 bp; The genomic location of SNP53 is Chr4: 166283036 bp; The genomic location of SNP54 is Chr4: 166283267 bp; The genomic location of SNP55 is Chr4: 166283416 bp; The genomic location of SNP56 is Chr4: 166283641 bp; The genomic location of SNP57 is Chr4: 166283653 bp; The genomic location of SNP58 is Chr4: 166283818 bp; The physical location information of the 58 SNP molecular markers was determined based on the maize B73 RefGen_v4 genome; When the haplotype sequence formed by SNP1 to SNP58 is as shown in SEQ ID NO.1, the flowering period of maize is the early flowering phenotype.

2. A method for identifying or assisting in the identification of the flowering period of maize, characterized in that, This includes detecting the genotype of the SNP molecular marker combination described in claim 1 in a maize sample to be tested, and identifying or assisting in the identification of the flowering period of maize based on the genotype of the maize sample to be tested; When the haplotype sequence formed by SNP1 to SNP58 is as shown in SEQ ID NO.1, the flowering period of maize is an early flowering phenotype; The corn variety is at least one of the following: corn B73, corn W22, corn B104, corn Chang 7-2, corn Dan 340, corn Qi 319, corn Ye 478, and corn Zheng 58.

3. The method according to claim 2, characterized in that, The maize in question is a maize inbred line.

4. The application of the method according to claim 2 or 3 in maize breeding; When the haplotype sequence formed by SNP1 to SNP58 is as shown in SEQ ID NO.1, the flowering period of maize is an early flowering phenotype; The corn variety is at least one of the following: corn B73, corn W22, corn B104, corn Chang 7-2, corn Dan 340, corn Qi 319, corn Ye 478, and corn Zheng 58.

5. The application according to claim 4, characterized in that, The maize breeding mentioned refers to selection and breeding during the maize flowering period.

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