Primer combination of molecular marker of ZmDFOT1 gene for regulating and controlling daily flowering time of corn and application of primer combination

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a primer combination of a molecular marker of a ZmDFOT1 gene for regulating and controlling the daily flowering time of corn and application of the primer combination. The primer combination consists of a PCR (Polymerase Chain Reaction) primer pair 1 and a PCR primer pair 2, the nucleotide sequence of the PCR primer pair 1 is as shown in SEQ ID NO. 3 to SEQ ID NO. 4; and the nucleotide sequence of the PCR primer pair 2 is as shown in SEQ ID NO.5-6. The nucleotide sequence of the ZmDFOT1 gene is as shown in SEQ ID NO. 1. The excellent haplotype of the corn daily flowering time regulating gene ZmDFOT1 can be used for identifying and screening materials with early daily flowering time of corn, and is suitable for cultivating new varieties of corn with early daily flowering time and high temperature resistance.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and genetic engineering, specifically relating to a method for regulating the daily flowering time of maize. ZmDFOT1 Primer combinations for molecular markers of genes and their applications. Background Technology

[0002] With global climate change, high temperatures have become one of the important abiotic stress factors affecting maize yield. It is predicted that every 1°C increase in average temperature will lead to a 7.4% reduction in maize yield. The impact of high temperatures on maize is mainly concentrated during the ear differentiation stage and the pollen and silking stage. In the Huang-Huai-Hai region, the summer maize growing season is characterized by simultaneous rain and heat, and the pollen and silking stage often coincides with high temperatures. This often causes the opening of maize tassels and spikelets to be hindered, resulting in a decrease in anther dehiscence rate and pollen leakage rate, reduced pollen viability and inability to fertilize, ultimately leading to a decrease in seed setting rate and yield.

[0003] Addressing the impact of high temperatures during the flowering period on maize production is a crucial need for ensuring maize yield and national food security. Currently, the main approach is to improve maize's heat resistance by utilizing heat-resistant genetic resources. However, with extreme heat events becoming increasingly frequent and poor ventilation in field maize fields, strategies to improve maize's heat resistance through heat-resistant methods cannot completely eliminate the adverse effects of high temperatures on maize production.

[0004] Diurnal Flower Opening Time (DFOT) refers to the time during which the spikelets open within a day. By advancing the opening time of maize spikelets, high temperatures can be avoided, promoting opening at lower temperatures to complete anther dehiscence and pollen shedding, thereby improving pollen viability and ear seed setting rate. Utilizing early-flowering germplasm resources to avoid high temperatures is an effective measure to address heat damage in maize. To achieve efficient and precise screening for the early-flowering trait, it is urgent to develop specific molecular marker primer combinations targeting key genes regulating this trait, enabling efficient and precise marker-assisted selection. Summary of the Invention

[0005] To develop specific molecular marker primer combinations for key genes regulating the daily flowering time trait in maize, this invention develops a specific molecular marker primer combination for key genes regulating the daily flowering time trait in maize, achieving efficient and precise screening for this trait. This invention develops molecular markers and primers for identifying superior haplotypes of the daily flowering time gene, thereby identifying germplasm resources with early daily flowering time. This invention provides a method for regulating the daily flowering time of maize. ZmDFOT1 Primer combinations for gene molecular markers and their applications. To achieve the above objectives and solve the aforementioned technical problems, the present invention adopts the following technical solution.

[0006] To identify key genes for daily flowering time and subsequently develop germplasm resources with early daily flowering time to address heat damage during maize flowering, this invention provides a method for regulating daily flowering time in maize. ZmDFOT1 A primer pair for a molecular marker of a gene, the primer pair consisting of PCR primer pair 1 and PCR primer pair 2.

[0007] The nucleotide sequences of the PCR primer pair 1 are shown in SEQ ID NO.3 (SNP1-2F: 5'-CCAAGCACCTTCTCACAAGT-3') and SEQ ID NO.4 (SNP1-2R: 5'-GACGCATCATTTGTGTAGCG-3').

[0008] The nucleotide sequences of the PCR primer pair 2 are shown in SEQ ID NO.5 (SNP3F: 5'-TGATCAACCGATGGGCATAC-3') and SEQ ID NO.6 (SNP3R: 5'-AGATATCACCTGGCAGAGCA-3').

[0009] The ZmDFOT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0010] To develop specific molecular marker primer combinations targeting key genes regulating the daily flowering time trait in maize, this invention develops a specific molecular marker primer combination for key genes regulating the daily flowering time trait in maize, achieving efficient and precise screening for this trait. This invention clones and functionally identifies genes regulating the daily flowering time of maize. ZmDFOT1 This invention provides a core molecular target for addressing the problem of high-temperature heat damage during maize flowering. High-temperature heat damage during maize flowering mainly occurs during the morning high-temperature period, leading to pollen shedding obstacles, decreased pollen viability, and pollination failure. Advancing the flowering time (i.e., concentrating flowering and pollen shedding during the early morning low-temperature window) is a key strategy to avoid this stress. This invention discloses for the first time... ZmDFOT1 Genes regulate the flowering time of maize throughout the day, and have been developed. ZmDFOT1 Primer combinations for molecular markers of superior haplotypes. This invention provides two sets of specific PCR primer combinations that can target and amplify key sites in genes regulating maize flowering time, transforming gene function research results into a practical tool that can be directly used for molecular marker-assisted selection. The use of dual primer combinations achieves a technological breakthrough from phenotypic identification to genotype screening, providing efficient and convenient molecular marker products for large-scale, high-throughput, and precise screening of early flowering traits in breeding materials.

[0011] Preferably, PCR primer pair 1 is a primer pair for amplifying SNP1 and SNP2, and PCR primer pair 2 is a primer pair for amplifying SNP3.

[0012] The SNP1 is located in the nucleotide sequence shown in SEQ ID NO.1. ZmDFOT1 The 5066th position of the gene has a base of either G or A.

[0013] The SNP2 is located in the nucleotide sequence shown in SEQ ID NO.1. ZmDFOT1 At position 4531 of the gene, the base is either C or T.

[0014] The SNP3 is located in the nucleotide sequence shown in SEQ ID NO.1. ZmDFOT1 The 2478th position of the gene has a base of either G or A.

[0015] The present invention also provides a kit for identifying the daily flowering time trait of maize, comprising the primer combination described above.

[0016] The present invention also provides the application of the primer combination or the kit in identifying the daily flowering time trait of maize.

[0017] Preferably, the steps for identifying the daily flowering time trait of maize are as follows: Genomic DNA was extracted from the maize material to be tested.

[0018] The genomic DNA was amplified by PCR using PCR primer pair 1 and PCR primer pair 2.

[0019] Analyze the PCR amplification product sequence to determine the presence of [specific components] in the maize material being tested. ZmDFOT1 The genotypes at positions 5066, 4531, and 2478 of the gene were used to identify the maize's daily flowering time trait.

[0020] Preferably, if the corn material to be tested contains ZmDFOT1 If the genotypes at positions 5066, 4531, and 2478 of the gene are GG, CC, and GG, then the maize material being tested is identified as having an early flowering time trait. ZmDFOT1 If the genotypes at positions 5066, 4531, and 2478 of the gene are AA, TT, and AA, then the maize daytime flowering time trait of the maize material to be tested is identified as late daytime flowering time.

[0021] Among them, "early flowering time" refers to the time when corn reaches its peak pollen shedding period, which is 7:00 to 8:00 every day.

[0022] Late flowering time refers to the time when corn reaches its peak pollen shedding period, which is between 8:30 and 9:30 every day.

[0023] Preferably, by simultaneously detecting the corn material to be tested... ZmDFOT1 Genotypes at positions 5066, 4531, and 2478 of the gene were used to screen for maize materials with early flowering time, genotypes GG, CC, and GG.

[0024] Preferably, the early-flowering maize material is used to breed high-yielding maize hybrids that avoid high temperatures.

[0025] Compared with the prior art, the present invention has the following beneficial effects: To develop specific molecular marker primer combinations targeting key genes regulating the daily flowering time trait in maize, this invention provides a specific molecular marker primer combination for key genes regulating the daily flowering time trait in maize, enabling efficient and precise screening of this trait. This invention also provides a gene for regulating the daily flowering time of maize. ZmDFOT1 This invention utilizes cloning and functional identification of substances that regulate the daily flowering time of maize. ZmDFOT1 This invention provides a core molecular target for addressing the problem of high-temperature heat damage during maize flowering. High-temperature heat damage during maize flowering mainly occurs during the morning high-temperature period, leading to pollen shedding obstacles, decreased pollen viability, and pollination failure. Advancing the flowering time (i.e., concentrating flowering and pollen shedding during the early morning low-temperature window) is a key strategy to avoid this stress. This invention discloses for the first time... ZmDFOT1 Genes regulate the flowering time of maize throughout the day, and have been developed. ZmDFOT1 Primer combinations for molecular markers of superior haplotypes. This invention provides two sets of specific PCR primer combinations that can target and amplify key sites of key genes regulating the daytime flowering trait in maize, transforming gene function research results into a practical tool that can be directly used for molecular marker-assisted selection. The use of dual primer combinations achieves a technological breakthrough from phenotypic identification to genotype screening, providing efficient and convenient molecular marker products for large-scale, high-throughput, and precise screening of early-flowering traits in breeding materials.

[0026] Meanwhile, the method for regulating the daily flowering time of maize provided by this invention... ZmDFOT1 Primer combinations of gene molecular markers can be used to screen for maize materials with early flowering time, advance the daily flowering time of maize, and enable maize to successfully complete the flowering, pollination and fertilization process before the arrival of high environmental temperatures, thereby improving pollen viability and ear seed setting rate. Attached Figure Description

[0027] Figure 1 This is a frequency distribution histogram of the daily flowering timetable data in this invention, wherein: A represents the environment of Hebi; B represents the environment in Zhangye.

[0028] Figure 2 For the genome-wide association analysis of the maize daily flowering time typology in this invention, wherein: A is the Manhattan map of the environment in Hebi; B is a QQ image of the environment in Hebi; C is the Manhattan diagram of Zhangye's environment; D is a QQ image of Zhangye's environment.

[0029] Figure 3 for ZmDFOT1 Gene biological functions, including: A is a mutant. dfot1 The identification; B represents wild-type and mutant. dfot1 Images of flowers blooming in the sun; C represents wild type and mutant. dfot1 Statistics on daily flowering time.

[0030] Figure 4 The BLUP of the Hebi and Zhangye environments in this invention ZmDFOT1 Association analysis, in which: A is the Manhattan diagram; B is a QQ image; C is ZmDFOT1 Scatter plot of correlation analysis; D is ZmDFOT1 Linkage disequilibrium analysis of gene regions, using R 2 A heatmap drawn based on this.

[0031] Figure 5 For the present invention ZmDFOT1 Comparison of daily flowering time by genotype, including: A represents the environment of Hebi; B represents the environment in Zhangye. ** P <0.01 (t-test). Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0033] Example 1 1. Analysis of daily flowering timeline data of related groups 1.1 Test Materials The materials used in this invention consist of a related population of 198 temperate maize inbred lines. These materials were all provided by Professor Yan Jianbing's research group at Huazhong Agricultural University.

[0034] 1.2 Field Trial Design The associated populations were planted in two environments: the experimental base of Hebi Academy of Agricultural Sciences, Hebi City, Henan Province (35°42′N, 114°19′E) in 2023, and Runfengyuan Agricultural Development Co., Ltd., Zhangye City, Gansu Province (39°6′N, 100°10′E) in 2024. A randomized block design was used, with two replicates in each environment. Each replicate consisted of one row, 4 m long, with a row spacing of 60 cm, and a density of 67,500 plants / hm². 2 Field management follows standard field management practices.

[0035] 1.3 Survey of daily flowering time patterns The maize plant as the observation unit was the individual maize plant from the aforementioned related population. After the maize entered the tasseling stage, the development of the tassel on each maize plant was closely monitored every morning. When 15 anthers emerged on the maize tassel, the time point was recorded; this time point was the daily flowering time of that individual plant.

[0036] 1.4 Data Processing The daily flowering time data of individual plants were converted into numerical format using Microsoft Excel 2021. To ensure data reliability, box plots were first created using IBM SPSS software to initially remove outliers. Then, outliers were further removed using the mean ± 1.5 times the standard deviation, and the average daily flowering time of the inbred lines was calculated for subsequent analysis.

[0037] Descriptive statistical analysis was performed on the daily flowering time time typographic data using Microsoft Excel 2021 and IBM SPSS software, including mean, maximum, minimum, standard deviation, skewness, and kurtosis. The best linear unbiased prediction (BLUP) for the Hebi and Zhangye environments was calculated using the R language (R Core Team) lme4, and the generalized heritability of daily flowering time was calculated using the following formula:

[0038] H 2 =δg 2 / (δg 2 +δe 2 / nr); In the above formula, δg 2 For the genetic variance, δe 2 Let be the error variance, n be the number of environments, and r be the number of repetitions.

[0039] Frequency distribution histograms of daily flowering time pattern data were plotted using Origin 2021 software for each environment.

[0040] 1.5 Research Results By investigating the daily flowering time phenotypic data in two environments (Hebi and Zhangye), it was found that the average daily flowering time of this associated population was 7:57 in the Hebi environment, with a variation range of 6:17 to 9:19; and 9:46 in the Zhangye environment, with a variation range of 8:01 to 11:04, indicating that the daily flowering time trait of this associated population exhibits relatively rich phenotypic variation (Table 1). The absolute values ​​of skewness and kurtosis of the daily flowering time trait in both environments were less than 1 (Table 1), and the data distribution curves conformed to a normal distribution. Figure 1 The results indicate that the daily flowering time trait in maize conforms to quantitative trait characteristics. The heritability of the daily flowering time trait is 0.62, indicating that in addition to genetic factors, the environment also plays an important role in this trait.

[0041] Table 1. Descriptive statistics of daily flowering timetable data. 2. Genome-wide association analysis of the daily flowering time trait in maize Genotypic data for the associated populations were downloaded from the website (http: / / www.maizego.org / Resources.html). This data contained 1,253,814 high-quality SNPs covering the entire maize genome with a minimum allele frequency ≥0.05. Phenotypic genome-wide association analysis was performed using Tassel 3.0 software with a general linear model. P ≤1.0×10 -5 This serves as a criterion for determining a significant association between SNPs and daily flowering time patterns.

[0042] A 50kb physical interval upstream and downstream of a significant SNP locus is defined as a quantitative trait locus. Referring to the reference genome of the maize inbred line B73 (B73RefGen_V4), functional annotation and gene sequence analysis were performed on candidate genes within the QTL intervals using the MaizeGDB database (http: / / www.maizegdb.org / gbrowse).

[0043] Genome-wide association analysis was performed on maize daily flowering time typographic data from two environments using a Q-model. P ≤1.0×10 -5 Significant associations were identified between the markers and traits. In the Hebi environment, 51 SNPs significantly associated with the daily flowering time trait of maize were detected, and in the Zhangye environment, 36 SNPs significantly associated with the daily flowering time trait of maize were detected. Among these, two SNPs located at chromosome 8 were repeatedly detected in both environments, indicating that these two SNPs are significantly associated with the daily flowering time of maize. Figure 2 ).

[0044] Based on linkage disequilibrium decay distance, three candidate genes were screened within a 50kb range upstream and downstream of two stable SNPs, among which... Zm00001d008648 Encoding zinc finger transcription factors, Zm00001d008649 Encoding E3 ligase, Zm00001d008650 Functionality unknown. Both stable SNPs are located in... Zm00001d008648 Genetically.

[0045] The results of the analysis of candidate genes for daily flowering time are shown in Table 2.

[0046] Table 2. Analysis of candidate genes for flowering time. Note: chr8.s_15871735 refers to the molecular marker at position 15871735 on chromosome 8; chr8.s_15872270 refers to the molecular marker at position 15872270 on chromosome 8.

[0047] 3. ZmDFOT1 Gene function verification To confirm Zm00001d008648 The biological function of genes regulating the daily flowering time of maize was obtained from the EMS mutant library (http: / / maizeems.qlnu.edu.cn / ). Zm00001d008648 The mutant gene (EMS4-15204c) was used. DNA was extracted from the leaves of the mutant plants using the SLS method. Using the leaf genomic DNA as a template, PCR amplification was performed using primers 8648F and 8648R. The PCR products were then subjected to agarose gel electrophoresis, gel extraction, and sequencing by Beijing Qingke Biotechnology Co., Ltd. to detect the mutation sites in the mutant.

[0048] The nucleotide sequence of primer 8648F is shown in SEQ ID NO.9: 5'-AGATTACCCGGCATTCAAGC-3'.

[0049] The nucleotide sequence of primer 8648R is shown in SEQ ID NO.10: 5'-ATTTTGCTTCTTAACGCCCA-3'.

[0050] The PCR amplification system (30 μL) consists of: 2 μL DNA, 3 μL primers (1.5 μL each for upstream and downstream primers), 15 μL 2×Phanta Max Buffer, 0.6 μL dNTP Mix, 0.6 μL Phanta Max Super-Fidelity DNA Polymerase, and 8.8 μL ddH2O.

[0051] PCR amplification program: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 30 s, for a total of 35 cycles; 72℃ for 5 min.

[0052] Sequencing results showed that, compared with the wild-type B73 genome sequence, the mutant had... Zm00001d008648 A C-to-T mutation occurred in the fifth exon of the gene, causing premature termination of translation. Figure 3 A). Daily flowering time typological analysis revealed that, compared to the wild type, the mutant's daily flowering time was significantly earlier (in section A). Figure 3 B and Figure 3 The C in the figure indicates that this gene is a key gene regulating the daily flowering time of maize (named C). ZmDFOT1 ).

[0053] According to predictions from MaizeGDB (https: / / maizegdb.org / ), Zm00001d008648 The gene has a nucleotide sequence length of 5271 bp and encodes a zinc finger transcription factor consisting of 757 amino acids.

[0054] in, Zm00001d008648 ( ZmDFOT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1:

[0055] Zm00001d008648 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2: MPSSYAAAAAGSSSRKPNRTSSAAAPTLRPPAPSPSPAPAPLAVNPSVISDSDPSSYSSSSADEADFAASDSATASVVSAYLSVAGEGADLSKVGIFLSSAARRRSPPCLICFDPIRPSDPVWSCSSSCFAILHLPCIQSWAHQSASGPAVPCPTWGCPKCRFAYPKSEIPSSYVCFCSKTIDPAPDPWILPHSCGDVCGRRLDANLDSGCEHTCLLLCHPGPCPPCPAVVPKATCFCGVHREPRRCAHQRYSCGGKCNKRLSCGLHHCPVDCHDGPCPPCAVLGSHKCECGEILEEKLCSERIFQCKRECGGMLDCGKHKCERGCHGGKCGECPLRGRRTCPCGKKNYPRLECDAEAATCGSTCEKVLGCGRHRCPERCHRGPCDGTCRLVVTKACRCGGLKKEVPCYQELTCERKCQRLRNCGRHACKRRCCAGDCPPCSETCDKKLRCGNHKCLSPCHRGACSPCPLMKTISCACGKTCFEVEFALLQVPCGTEKNQKPPKCSKKCNIPRLCRHKLECRPHKCHYGACPPCKLICGEEFPCGHTCKERCHGPISPPNSEFTLKPTKKKMGKHIECTPGTPCPPCKEVVLVSCFGQHLGQERTVNTRCACSTLKQEWRCQDVLKEYRKSGRDPKEVPKSQFGVGLLACGEDCKKKLKAPDSELHLRKSQENKIPAVEVVNAPKRRKRRDRGQEVKISKFQEVKTFVLRALLIILLSIIVAAGLYLLWKGIFWLSDWMNEMEEQRASQRYPRGAML.

[0056] Zm00001d008648 The nucleotide sequence of the mutant of the gene (EMS4-15204c) is shown in SEQ ID NO.7:

[0057] Zm00001d008648 The amino acid sequence of the mutant gene (EMS4-15204c) is shown in SEQ ID NO.8: MPSSYAAAAAGSSSRKPNRTSSAAAPTLRPPAPSPSPAPAPLAVNPSVISDSDPSSYSSSSADEADFAASDSATASVVSAYLSVAGEGADLSKVGIFLSSAARRRSPPCLICFDPIRPSDPV WSCSSSCFAILHLPCIQSWAHQSASGPAVPCPTWGCPKCRFAYPKSEIPSSYVCFCSKTIDPAPDPWILPHSCGDVCGRRLDANLDSGCEHTCLLLCHPGPCPPCPAVVPKATCFCGVHREPR RCAHQRYSCGGKCNKRLSCGLHHCPVDCHDGPCPPCAVLGSHKCECGEILEEKLCSERIFQCKRECGGMLDCGKHKCERGCHGGKCGECPLRGRRTCPCGKKNYPRLECDAEAATCGSTCEKV LGCGRHRCPERCHRGPCDGTCRLVVTKACRCGGLKKEVPCYQELTCERKCQRLRNCGRHACKRRCCAGDCPPCSETCDKKLRCGNHKCLSPCHRGACSPCPLMKTISCACGKTCFEVEFALL.

[0058] 4. Candidate gene association analysis ZmDFOT1 Genotype data for gene association analysis were downloaded from the website (http: / / www.maizego.org / Resources.html). Extraction ZmDFOT1 A new genotype file was constructed by extracting the full-length gene and SNPs in the 2000bp region upstream of the start codon. BLUP data on flowering time from two environmental days were integrated with the extracted candidate gene genotype files, and association analysis of the candidate genes was performed using Tassel 3.0 software. Furthermore, linkage disequilibrium heatmaps of the candidate gene regions were generated using LDBlockShow software.

[0059] Using the Q-model, with a threshold of 5.7, SNPs significantly associated with daily flowering time were identified at the anterior end of chromosome 8. Among them, three SNPs significantly associated with daily flowering time were located at... ZmDFOT1 Genetically, they are chr8.s_15872270 ( P =4.82×10-10 ), chr8.s_15871735 ( P =1.27×10 -9 ), chr8.s_15869682 ( P =6.69×10 -9 () Figure 4 ).

[0060] Among them, chr8.s_15872270 is SNP1, chr8.s_15871735 is SNP2, and chr8.s_15869682 is SNP3.

[0061] SNP1 is located in the nucleotide sequence shown in SEQ ID NO.1. ZmDFOT1 The 5066th position of the gene has a base of either G or A.

[0062] SNP2 is located in the nucleotide sequence shown in SEQ ID NO.1. ZmDFOT1 At position 4531 of the gene, the base is either C or T.

[0063] SNP3 is located in the nucleotide sequence shown in SEQ ID NO.1. ZmDFOT1 The 2478th position of the gene has a base of either G or A.

[0064] 5. ZmDFOT1 Gene haplotype analysis Extracted from genome-wide association analysis results ZmDFOT1 Genotype data of significant SNPs were used to segment associated populations using Excel software. ZmDFOT1 Genetic haplotype. Analyzed using a t-test. ZmDFOT1 The difference in flowering time between different haplotypes was investigated, and phenotypic data were plotted using Origin software.

[0065] in accordance with ZmDFOT1 Three significant SNPs detected by BLUP association analysis of flowering time on two environmental days were found in the natural population. ZmDFOT1 Genes are classified into haplotypes, with two main types: Hap1 (GCG) and Hap2 (ATA) (Table 3). For identification... ZmDFOT1 The superior haplotypes were used to analyze the differences in daily flowering time between two haplotypes in the Hebi and Zhangye environments. In the Hebi environment, Hap1 contained 132 inbred lines, and Hap2 contained 12 inbred lines; in the Zhangye environment, Hap1 contained 169 inbred lines, and Hap2 contained 14 inbred lines. Statistical analysis showed that the daily flowering time of Hap1 was significantly earlier than that of Hap2 in both the Hebi and Zhangye environments. P <0.01)( Figure 5).therefore, ZmDFOT1 The gene Hap1 (GCG) is ZmDFOT1 A superior haplotype that regulates the daily flowering time of maize.

[0066] Table 3 ZmDFOT1 Gene haplotype 6. ZmDFOT1 Application of gene / molecular markers DNA was extracted from maize plant leaves using the SLS method. Using leaf genomic DNA as a template, PCR amplification was performed using primer pairs SNP1-2F, SNP1-2R, and SNP3F and SNP3R. The PCR amplification products were then subjected to agarose gel electrophoresis, gel extraction, and sequencing by Beijing Qingke Biotechnology Co., Ltd. to determine the genotype of maize plant leaves.

[0067] Among them, the maize plant was ZHENG35, which was derived from a related population provided by Professor Yan Jianbing's research group at Huazhong Agricultural University.

[0068] The detection primers for SNP1 and 2 are SNP1-2F and SNP1-2R. The nucleotide sequence of SNP1-2F is shown in SEQ ID NO.3. 5'-CCAAGCACCTTCTCACAAGT-3'.

[0069] The nucleotide sequence of SNP1-2R is shown in SEQ ID NO.4: 5'-GACGCATCATTTGTGTAGCG-3'.

[0070] The SNP3 detection primers are SNP3F and SNP3R. The nucleotide sequence of SNP3F is shown in SEQ ID NO.5. 5'-TGATCAACCGATGGGCATAC-3'.

[0071] The nucleotide sequence of SNP3R is shown in SEQ ID NO.6: 5'-AGATATCACCTGGCAGAGCA-3'.

[0072] The PCR amplification system (30 μL) consists of: 2 μL DNA, 3 μL primers (1.5 μL each for upstream and downstream primers), 15 μL 2×Phanta Max Buffer, 0.6 μL dNTP Mix, 0.6 μL Phanta Max Super-Fidelity DNA Polymerase, and 8.8 μL ddH2O.

[0073] PCR amplification program: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 2 min 30 s, for a total of 35 cycles; 72℃ for 5 min.

[0074] Based on the above genotype results, determine (identify) whether the corn plant (single plant) has the ability to flower early in the day.

[0075] Using the B73-V4 version of the genome as a reference, when there is a G base at position 15872270, a C base at position 15871735, and a G base at position 15869682 on chromosome 8, the daily flowering time of the maize plant is earlier.

[0076] As shown above, the SNP1-2F / SNP1-2R and SNP3F / SNP3R primer pairs can be used to determine whether a maize plant is an early-flowering individual. If the genotypes at chromosome 8 at positions 15872270, 15871735, and 15869682 are GG, CC, and GG, then the maize plant is an early-flowering maize plant; if they are AA, TT, and AA, then the maize plant is a late-flowering maize plant.

[0077] This invention investigated the daily flowering time typology of three Hap1 inbred lines and seven Hap2 inbred lines, finding that the daily flowering time of the Hap1 inbred lines was significantly earlier than that of the Hap2 inbred lines (see Table 4). The three Hap1 and seven Hap2 inbred lines were derived from a maize-related population provided by Professor Yan Jianbing's research group at Huazhong Agricultural University.

[0078] Table 4 Comparison of daily flowering time of different haplotype inbred lines In summary, this invention regulates the daily flowering time of maize by cloning and functionally identifying [a specific mechanism / method]. ZmDFOT1 This invention provides a core molecular target for addressing the problem of high-temperature heat damage during maize flowering. High-temperature heat damage during maize flowering mainly occurs during the morning high-temperature period, leading to pollen shedding obstacles, decreased pollen viability, and pollination failure. Advancing the flowering time (i.e., concentrating flowering and pollen shedding during the early morning low-temperature window) is a key strategy to avoid this stress. This invention discloses for the first time... ZmDFOT1 Genes regulate the flowering time of maize throughout the day, and have been developed. ZmDFOT1Primer combinations using molecular markers of superior haplotypes enable precise modification of maize daily flowering time through marker-assisted selection. Utilizing these superior haplotype markers, maize germplasm resources with early flowering times can be efficiently identified. This allows the flowering and pollen shedding process to proactively avoid peak high-temperature periods, completing pollination during relatively cooler times. This effectively ensures anther dehiscence, pollen viability, and silk fertilization capacity, ultimately mitigating the negative impact of high-temperature heat damage during flowering on maize seed setting rate and yield. This enables the targeted creation and application of stress-resistant germplasm, solving the problem of precise phenotypic identification in existing technologies where the daily flowering time trait is highly susceptible to environmental influences.

[0079] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

[0080] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0081] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. Regulating the daily flowering time of maize ZmDFOT1 Primer combinations for gene molecular markers, characterized in that, The primer pair consists of PCR primer pair 1 and PCR primer pair 2; The nucleotide sequences of the PCR primer pair 1 are shown in SEQ ID NO.3~SEQ ID NO.4; The nucleotide sequences of the PCR primer pair 2 are shown in SEQ ID NO.5~SEQ ID NO.6; The ZmDFOT1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The primer combination according to claim 1, characterized in that, The PCR primer pair 1 is a primer pair used to amplify SNP1 and SNP2, and the PCR primer pair 2 is a primer pair used to amplify SNP3. The SNP1 is located in ZmDFOT1 At position 5066 of the gene, the base is either G or A; The SNP2 is located in ZmDFOT1 At position 4531 of the gene, the base is either C or T; The SNP3 is located in ZmDFOT1 The 2478th position of the gene has a base of either G or A.

3. A kit for identifying the daily flowering time trait of maize, characterized in that, Includes the primer combination described in any one of claims 1 to 2.

4. The use of the primer combination described in any one of claims 1 to 2 or the kit described in claim 3 in identifying the daily flowering time trait of maize.

5. The application according to claim 4, characterized in that, The steps for identifying the daily flowering time trait in maize are as follows: Genomic DNA was extracted from the maize material to be tested; The genomic DNA was amplified by PCR using PCR primer pair 1 and PCR primer pair 2; Analyze the PCR amplification product sequence to determine the presence of [specific components] in the maize material being tested. ZmDFOT1 The genotypes at positions 5066, 4531, and 2478 of the gene were used to identify the maize's daily flowering time trait.

6. The application according to claim 5, characterized in that, If the corn material to be tested contains ZmDFOT1 If the genotypes at positions 5066, 4531, and 2478 of the gene are GG, CC, and GG, then the maize material being tested is identified as having an early flowering time trait. ZmDFOT1 If the genotypes at positions 5066, 4531, and 2478 of the gene are AA, TT, and AA, then the maize daytime flowering time trait of the maize material to be tested is identified as late daytime flowering time. Among them, "early flowering time" refers to the time when corn reaches its peak pollen shedding period, which is 7:00-8:00 every day; Late flowering time refers to the time when corn reaches its peak pollen shedding period, which is between 8:30 and 9:30 every day.

7. The application according to claim 6, characterized in that, By simultaneously detecting the corn material under test ZmDFOT1 Genotypes at positions 5066, 4531, and 2478 of the gene were used to screen for maize materials with early flowering time, genotypes GG, CC, and GG.

8. The application according to claim 7, characterized in that, The corn materials with early flowering time are used to breed high-yield corn hybrids that avoid high temperatures.

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