A maize photothermal sensitive male sterility gene mutant and application thereof

Abstract

This invention relates to the field of maize molecular breeding technology, specifically disclosing a photoperiod-sensitive male sterility gene mutant in maize and its application. The mutant is obtained by a single-base mutation in the KO1 gene (Zm00001d046344 gene in the MaizeGDB database, reference genome RefGen_v4 version of inbred line B73), specifically mutants ko1-4, ko1-19, or ko1-22, with mutation sites at positions 6329, 5821, and 5659 from the start codon, respectively, where G is mutated to A. Maize carrying this mutant gene exhibits complete male sterility under long-day, high-temperature conditions, suitable for hybrid seed production; under short-day, low-temperature conditions, the male tassels are fully fertile, allowing for self-pollination, thus achieving a "one-line, two-use" two-line male sterility seed production method.

Description

Technical Field

[0001] This invention belongs to the field of maize molecular breeding technology, specifically relating to a photoperiod-temperature-sensitive male sterility gene mutant in maize and its application. Background Technology

[0002] Maize is the world's largest food crop and an important source of feed and bioenergy. Utilizing heterosis is a key technology for improving maize yield and stress resistance. In hybrid seed production, the efficiency and purity of female parent emasculation directly affect the quality and cost of hybrid seeds. Traditional manual and mechanical emasculation methods are not only labor-intensive and costly, but also cannot guarantee 100% emasculation purity, posing a risk of self-pollination of the female parent. Therefore, using male-sterile lines for hybrid seed production has become an important direction in maize breeding.

[0003] Currently, maize male sterility is mainly divided into two categories: cytoplasmic male sterility (CMS) and genic male sterility (GMS). CMS has been widely used in commercial hybrid production, such as T, C, and S cytoplasmic sterile types. However, CMS has significant limitations: first, sterility is easily affected by environmental conditions, and partial recovery of fertility may occur under high temperature or specific light conditions, leading to seed production failure; second, the cytoplasmic source is singular, making it prone to specialized diseases. For example, T-type CMS caused a large-scale reduction in maize yield in the United States due to the outbreak of Cochliobolus heterostrophus T race; third, the recovery gene resources of CMS are limited, restricting the formulation of strong-influence hybrid combinations. While traditional GMS has stable sterility and is not affected by the environment, it usually cannot be propagated through self-pollination and needs to be maintained by linkage markers or semi-sterile plants, resulting in high propagation costs and low efficiency, making it difficult to apply on a large scale to seed production.

[0004] To overcome the shortcomings of the two aforementioned systems, photo-thermo-sensitive genic male sterility (PTGMS) technology was developed. PTGMS is a type of nuclear male sterility regulated by photoperiod and / or temperature: under specific photo-temperature conditions (such as long-day, high-temperature), the plant exhibits male sterility and can be used for hybrid seed production; under other photo-temperature conditions (such as short-day, low-temperature), the plant regains fertility and can self-pollinate. This "two-line method" for utilizing heterosis combines the advantages of CMS and GMS, avoiding negative cytoplasmic effects and disease risks while solving the problem of male-sterile line propagation. This technology has achieved great success in rice, such as the breeding and application of photo-thermo-sensitive male-sterile lines like Anong S-1 and Peiai 64S, which has promoted the large-scale promotion of two-line hybrid rice.

[0005] However, the discovery and utilization of photoperiod- and temperature-sensitive male sterile resources in maize remains severely lagging. Currently, only a few photoperiod- and temperature-sensitive male sterile materials in maize have been reported, such as "Qiong 42ms" and "C815S". These materials typically have the following problems: First, sterility is controlled by multiple quantitative trait loci (QTLs), the genetic mechanism is complex, the critical photoperiod and temperature thresholds for fertility conversion are unstable, and there are large differences in performance between different years and locations, making it difficult to reliably apply them to commercial seed production; Second, the photoperiod and temperature response types of these materials are mostly "high temperature / long day fertile, low temperature / short day sterile" or the opposite type, which do not match the photoperiod and temperature conditions of the seed production season in the main maize producing areas well; Third, there is a lack of cloning and functional analysis of key genes in these materials, making it impossible to develop co-dominant molecular markers for efficient assisted selection. In addition, most of the maize male sterility genes that have been cloned so far (such as MS26, MS45, ZmMs1, ZmTGA9, etc.) are either completely sterile mutations or temperature-sensitive mutations that are only affected by temperature. They do not yet have the dual photothermal-sensitive conversion characteristics that simultaneously satisfy "sterile in seed production site and fertile in seed propagation site", and therefore cannot be directly used for two-line seed production.

[0006] A search revealed the following authorized invention patent: "A gene ZmKO1 regulating pollen shedding from male spikelets in maize and its application in male-sterile hybrid seed production in maize" (Patent No.: CN119307539B, Authorization Announcement Date: August 5, 2025, Patentee: Sichuan Agricultural University). The main content and implementation scheme of this prior art are as follows: This patent discloses a gene, ZmKO1 (gene number Zm00001eb385100, NCBI gene number 100274015), that regulates the flowering and pollen shedding of maize male spikelets. This gene encodes endoroot-kaurene oxidase 1, which participates in gibberellin synthesis. The inventors screened a male-sterile mutant, def1, from a maize mutagenesis population, characterized by inhibited filament elongation, failure of glumes to dehisce, and inability of anthers to exfoliate and release pollen. Genetic analysis showed that this mutation is controlled by a single recessive nuclear gene. Map-based cloning revealed a large insertion in the first exon of the ZmKO1 gene in the def1 mutant, leading to frameshift and premature termination of protein translation. The patent further used CRISPR-Cas9 technology to perform site-specific editing of the second exon of the ZmKO1 gene (target sequence 5'-GTCTCTGGTTTACCCCTGAT-3'), obtaining various editing types (such as 1bp insertion, 1bp deletion, and 16bp deletion), all resulting in male sterility.

[0007] The core feature of this existing technology is that the fertility of sterile lines can be restored by exogenously spraying gibberellins (such as GA3, GA1, GA4, GA7, or gibberellic acid). Specifically, under natural growth conditions, the ZmKO1 gene mutant exhibits male sterility and can be used as a female parent for hybrid seed production. When it is necessary to propagate sterile lines, spraying gibberellins on the male inflorescence during the silking stage allows the glumes to open normally, the anthers to be exposed and pollen to be released, thus enabling self-pollination. Based on this, the patent proposes a "two-line method" sterile hybrid seed production technology system: utilizing gibberellins as a chemical inducer for fertility restoration to achieve economical and efficient propagation of sterile lines, followed by hybridization seed production under natural conditions.

[0008] However, this existing technology has the following inherent shortcomings: 1. Reliance on exogenous hormone spraying increases costs and labor: To achieve self-pollination of sterile lines, gibberellin spraying must be applied to the male inflorescence during the silking stage. This not only increases the cost of pesticides (hormones) and manual application, but also requires precise control of the spraying timing and concentration, making the operation cumbersome and unsuitable for large-scale, mechanized seed production.

[0009] 2. Fertility recovery effect is affected by the environment: The induction effect of gibberellin may be affected by environmental conditions such as temperature and humidity, and there is a risk of incomplete recovery, which may lead to failure of self-pollination of sterile lines or reduced reproductive efficiency.

[0010] 3. Potential environmental safety and residue risks: Although gibberellin is a plant growth regulator, its frequent use in field production may still have an impact on the ecological environment and non-target organisms, and may trigger regulatory restrictions on hormone use.

[0011] 4. Most mutation types are loss-of-function frameshift mutations: The mutants of this existing technology (including def1 and CRISPR edited lines) are all complete loss-of-function mutations of the ZmKO1 gene. Their sterility is not sensitive to light and temperature and must rely on exogenous gibberellin to restore fertility, rather than using natural light and temperature conditions to achieve fertility conversion. Summary of the Invention

[0012] The purpose of this invention is to address the problems existing in the prior art by providing a photoperiod-temperature-sensitive male sterility gene mutant in maize and its application. The inventors constructed a nearly saturated maize EMS mutant library. In the early screening of gibberellin synthesis gene mutants, they discovered that three allelic mutants of the gibberellin synthesis pathway gene KO1 (gene number: Zm00001d046344) exhibited complete male sterility in Beijing environments and complete male fertility in Hainan environments, with no impact on plant architecture and yield traits in either environment. Therefore, these three allelic mutants of KO1 can be used as male-sterile lines for hybrid seed production in Beijing and as maintainer lines for self-pollination in Hainan. Compared with existing technologies, the mutants of this invention have significant advantages such as complete fertility conversion, stable environmental response, no impact on agronomic traits such as plant height and yield, and a simple genetic mechanism (single-gene control). Furthermore, this invention provides primer pair combinations, kits, and detection methods for specifically detecting these three mutation sites, enabling high-throughput genotyping identification and providing a novel gene resource and application scheme for male-sterile hybrid seed production in maize using the two-line method.

[0013] This invention provides the following technical solution: a mutant of the maize photoperiod-temperature-sensitive male sterility gene, wherein the mutant is obtained by a single-base mutation in the maize photoperiod-temperature-sensitive male sterility gene KO1, the gene number of the KO1 gene is Zm00001d046344, and the DNA sequence is shown in SEQ ID NO: 1; the mutant is mutant ko1-4 or mutant ko1-19 or mutant ko1-22; The mutant ko1-4 was obtained by changing the base from G to A at position 6329 of the KO1 gene DNA sequence starting from the start codon; The mutant ko1-19 was obtained by changing the base from G to A at position 5821 of the KO1 gene DNA sequence, starting from the start codon. The mutant ko1-22 was obtained by changing the base from G to A at position 5659 of the KO1 gene DNA sequence, starting from the start codon.

[0014] A primer pair composition for detecting the above-mentioned photoperiod-temperature-sensitive male sterility gene mutant in maize, the primer pair composition comprising: a primer pair for specifically amplifying mutant ko1-4, a primer pair for specifically amplifying mutant ko1-19, or a primer pair for specifically amplifying mutant ko1-22; The primer pair for the specific amplification mutant ko1-4 or the primer pair for the specific amplification mutant ko1-19 consists of a forward primer F-1 and a reverse primer R-1. The nucleotide sequence of the forward primer F-1 is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer R-1 is shown in SEQ ID NO: 4. The primer pair for specifically amplifying the mutant ko1-22 consists of a forward primer F-2 and a reverse primer R-2. The nucleotide sequence of the forward primer F-2 is shown in SEQ ID NO: 5, and the nucleotide sequence of the reverse primer R-2 is shown in SEQ ID NO: 6.

[0015] The application of the above primer pair composition in the preparation of a kit for detecting photoperiod-temperature-sensitive male sterility gene mutants in maize.

[0016] A kit comprising the primer pairs described above for identifying mutant ko1-4, mutant ko1-19, or mutant ko1-22.

[0017] The above-mentioned photoperiod-temperature-sensitive male sterility gene mutants in maize are used in the breeding of dual-purpose male sterile lines.

[0018] Furthermore, the dual-purpose male-sterile maize line is a maize material whose male fertility can switch between male sterility and fertility under different growth environments.

[0019] The above-mentioned application of the maize photoperiod-sensitive male sterility gene mutant in the breeding of dual-purpose male sterile maize lines includes the following method: crossing a donor parent carrying a homozygous ko1-4, ko1-19, or ko1-22 mutant gene with a recipient inbred line to obtain the F1 generation; using the F1 generation as the maternal parent, backcrossing with the recipient inbred line for 3 to 5 consecutive generations; performing PCR amplification and sequencing of the maize photoperiod-sensitive male sterility gene mutant gene combination using primers in each generation; identifying the genotype of individual offspring plants based on the mutation site base (G / A); selecting heterozygous plants carrying the target mutant gene and whose genetic background is similar to that of the recipient parent for the next round of backcrossing; after the backcrossing is completed, self-pollination and molecular marker screening are performed to obtain a homozygous mutant dual-purpose male sterile line with agronomic traits consistent with the recipient inbred line.

[0020] A method for detecting a photoperiod-sensitive male sterility gene mutant in maize involves PCR amplification of the genomic DNA of the maize to be tested using the primer pair composition described above. After sequencing the PCR amplification product, the genotype of the photoperiod-sensitive male sterility gene carried by the maize to be tested is determined based on the bases at the mutation site. Maize plants carrying the mutant photoperiod-sensitive male sterility gene exhibit male sterility under specific light and temperature conditions.

[0021] Furthermore, the mutation site is selected from one of the following: The DNA sequence of the maize KO1 gene Zm00001d046344 has a mutation site at position 6329 from the start codon. The mutation site has a base G, indicating a wild-type genotype, and a mutation site has a base A, indicating a mutant genotype. The DNA sequence of the maize KO1 gene Zm00001d046344 has a mutation site at position 5821 from the start codon. The mutation site has a base G, indicating a wild-type genotype, and a mutation site has a base A, indicating a mutant genotype. The DNA sequence of the maize KO1 gene Zm00001d046344 has a mutation site at base 5659 from the start codon. The mutation site has a base G, indicating a wild-type genotype, and a mutation site has a base A, indicating a mutant genotype.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. No exogenous hormones are required; fertility is automatically regulated by natural light and temperature conditions. The fertility conversion of the three mutants in this invention relies entirely on natural photoperiod and temperature differences: under long-day, high-temperature environments (such as Beijing summer), the male tassels are completely sterile and can be directly used as female parents for hybrid seed production; under short-day, low-temperature environments (such as Hainan winter), the male tassels are completely fertile and can self-pollinate. The entire process requires no application of any exogenous hormones, eliminating the cost, labor, and technical aspects of gibberellin treatment, making it more economical, environmentally friendly, and convenient.

[0023] 2. The fertility conversion is thorough and stable, with no residual risk. Experimental data show that the mutant of this invention exhibits complete sterility (no pollen shedding in male spikes under long-day, high-temperature environments, such as Beijing summers) with no risk of self-pollination and can be used as a sterile line for hybrid seed production. Under short-day, low-temperature environments (such as Hainan winters), it is fully fertile and self-pollinates normally. The fertility conversion is thorough and clearly defined, unaffected by annual climate fluctuations, overcoming the problems of incomplete fertility conversion and critical condition drift in some existing sterile materials. It also overcomes the problem of unstable gibberellin induction in existing technologies. Furthermore, it avoids the potential risk of exogenous hormone residues in seeds and the environment.

[0024] 3. The genetic mechanism is simple, the molecular markers are precise, and it is easy to use in breeding. The three mutation sites in this invention are all single-base mutations controlled by a single gene, and their genetic behavior follows Mendelian laws, without involving complex multi-gene interactions. They are easily converted to male sex through backcrossing using molecular marker-assisted selection. Breeders can rapidly introduce photoperiod- and temperature-sensitive male sterility genes into target inbred lines to create maternally sterile lines through backcrossing, molecular marker-assisted selection, and other methods. The operation is simple and the cycle is short. This invention also provides primer pair compositions (SEQ ID NO: 3-6) for specific detection of these three mutation sites, enabling high-throughput and accurate genotyping identification and significantly improving breeding efficiency.

[0025] 4. Does not affect agronomic traits, ensuring seed production yield. Experimental data show that the key agronomic traits of mutants ko1-4, ko1-19, and ko1-22, such as plant height, ear height, and yield per plant, are not significantly different from those of wild-type B73. Therefore, the sterile lines created using these mutants will not introduce negative effects on yield or plant type and can be directly used for the sterilization improvement of superior inbred lines, ensuring the yield potential of hybrids.

[0026] 5. Provide complete reagent kits and application protocols. This invention not only provides primer pair compositions, but also a kit containing these primer pair compositions, as well as a complete technical solution based on the aforementioned primers and detection methods. This kit is easy to operate, has low detection costs, and is commercially viable. It provides a standardized tool for the identification, propagation, and seed production of photoperiod-sensitive male-sterile maize lines, and possesses significant commercial potential.

[0027] 6. Provides novel photoperiod-sensitive male sterility gene resources for maize. This invention reveals for the first time three novel single-base mutation sites—ko1-4, ko1-19, and ko1-22—in the maize KO1 gene (gene number: Zm00001d046344). These mutation sites can independently induce photoperiod- and temperature-sensitive male sterility in maize, greatly enriching the maize male sterility gene pool and providing new and usable sterility resources for two-line hybrid seed production in maize.

[0028] 7. Provide specific molecular markers and high-throughput detection methods. This invention designs specific PCR amplification primer pairs for three mutation sites (SEQ ID NO: 3 and SEQ ID NO: 4 for detecting mutant ko1-4 / ko1-19; SEQ ID NO: 5 and SEQ ID NO: 6 for detecting mutant ko1-22), and combines this with Sanger sequencing to achieve accurate determination of the mutant genotype. This method is highly specific and reproducible, and can efficiently distinguish between wild-type homozygous, mutant homozygous, and heterozygous genotypes, making it suitable for genotype screening of large-scale breeding materials.

[0029] 8. Expand the application ecological zones of the two-line seed production method for maize. The mutant strain of this invention performs excellently under the "Beijing summer seed production and Hainan winter seed propagation" model, making full use of the light and temperature differences between different ecological zones in northern and southern my country. It breaks through the limitations of existing temperature-sensitive male-sterile lines that are dependent on a single temperature factor and have critical temperature drift, providing a more stable and reliable solution for maize two-line hybrid seed production.

[0030] In summary, this invention upgrades the artificial hormone-induced fertility restoration mode to an automatic fertility conversion mode driven by natural light and temperature conditions, truly realizing the "dual use of one line" in maize two-line male-sterile hybrid seed production—sterile in the seed production field (high temperature, long days) and fertile in the seed propagation field (low temperature, short days). This invention provides entirely new genetic resources and a superior technical solution for maize male-sterile breeding. Attached Figure Description

[0031] Figure 1 This section presents phenotypic analyses of wild-type B73 and KO1 mutants under different environmental conditions. A represents the whole-plant phenotype in Beijing (scale bar = 50 cm); B represents plant height statistics in Beijing; C represents ear height statistics in Beijing; D represents the whole-plant phenotype in Hainan (scale bar = 30 cm); E represents plant height statistics in Hainan; F represents ear height statistics in Hainan; G represents the ear phenotype under open pollination conditions in Beijing (scale bar = 5 cm); H represents the yield statistics per plant under open pollination conditions in Beijing; I represents the ear phenotype under artificial self-pollination conditions in Hainan (scale bar = 5 cm); J represents the yield statistics per plant under artificial self-pollination conditions in Hainan; K represents the tassel phenotype in Beijing; and L represents the tassel phenotype in Hainan.

[0032] Figure 2 This data represents the temperature and light duration for 7 days before and after pollination / silking of different KO1 mutants planted in Sanya, Hainan in the winter of 2024 and in Beijing in the summer of 2025. A represents the daily maximum temperature before and after pollination; B represents the daily minimum temperature before and after pollination; C represents the daily average temperature before and after pollination; and D represents the daily light duration before and after pollination. The horizontal axis represents the 7 days before and after the start of pollination, with 0 representing the day pollination begins. Mutants in Beijing do not produce pollination; the time to silking is used for calculation.

[0033] Figure 3 These are Sanger sequencing results for different mutation sites in KO1. A represents the sequencing results of mutant ko1-4; B represents the sequencing results of mutant ko1-19; and C represents the sequencing results of mutant ko1-22. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0035] The gene number of the maize KO1 gene involved in the following embodiments of the present invention is Zm00001d046344, and its DNA sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2.

[0036] Example 1 The applicant constructed a nearly saturated maize EMS mutant library (http: / / maizeems.qlnu.edu.cn / ) using EMS mutagenesis and next-generation sequencing technologies. From this library, mutation sites related to the KO1 gene were screened, and three mutation sites were identified that cause complete male sterility in maize under specific environmental conditions. Specifically: The DNA sequence of the maize KO1 gene has a mutation site at position 6329 from the start codon. The mutation site has a base of G, indicating a wild-type genotype, and a mutation site has a base of A, indicating a mutant genotype. This mutant gene is named ko1-4. The single-base mutation causes the amino acid at position 475 to change from tryptophan W (codon TGG) to the stop codon (TGA).

[0037] The DNA sequence of the maize KO1 gene has a mutation site at position 5821 from the start codon. The mutation site has a base of G, indicating a wild-type genotype, and a base of A, indicating a mutant genotype. This mutant gene is named ko1-19. The single-base mutation causes the splice site receptor of the sixth intron to change from AG to AA, resulting in abnormal intron splicing.

[0038] The DNA sequence of the maize KO1 gene has a mutation site at position 5659 from the start codon. The mutation site has a base of G, indicating a wild-type genotype, and a base of A, indicating a mutant genotype. This mutant gene is named ko1-22. The single-base mutation causes the amino acid at position 310 to change from tryptophan W (codon TGG) to a stop codon (TGA).

[0039] Twenty seeds each of the mutant and wild-type B73 were selected and sown simultaneously in the experimental field during the normal growing seasons of summer in Beijing and winter in Hainan. Agronomic traits were investigated after all the mutant and wild-type seeds had shed pollen, and the phenotype of the ear was investigated after harvest. Figure 2 The data includes temperature and light exposure times for 7 days before and after pollination / silking of different KO1 mutants planted in Sanya, Hainan in the winter of 2024 and in Beijing in the summer of 2025.

[0040] The results are as follows Figure 1As shown, in both Beijing and Hainan environments, the plant architecture of the mutant ko1-4, ko1-19, and ko1-22 genotype maize was not significantly different from that of the wild-type B73 (plant height and ear height remained unchanged). In Beijing, the tassels of the wild-type B73 could release pollen normally, while the tassels of the mutant ko1-4, ko1-19, and ko1-22 genotypes did not release pollen at all. In Hainan, the tassels of both the wild-type B73 and the mutants ko1-4, ko1-19, and ko1-22 could release pollen normally. Under open pollination conditions, the yield per plant of the mutants ko1-4, ko1-19, and ko1-22 was not significantly different from that of the wild type.

[0041] In summary, all three mutation sites in KO1 result in sterility under specific conditions and do not affect plant type or yield-related traits. Therefore, any one of these three mutation sites in KO1 can be used to create a female-sterile line through backcrossing, enabling self-pollination for seed production in Hainan and hybrid seed production in Beijing. This makes it an ideal material for male-sterile hybrid seed production in maize.

[0042] Example 2 Using the genomic sequences flanking the mutation site in Example 1 as templates for primer synthesis, forward and reverse primers were designed to the left and right of the mutation site using Primer Premier5. Primer design principles: Tm value around 60℃, product size around 600 bp, and primer length 20-24 bp.

[0043] The primer pairs designed for each male sterility locus are as follows: Primer pairs specifically amplifying mutants ko1-4 or ko1-19 consist of forward primer F-1 and reverse primer R-1. Forward primer F-1: 5'-CCTGCTGGCAGAGAATACACTG-3' (SEQ ID NO: 3). Reverse primer R-1: 5'-GTGCGAGGTACACATGCAGTG-3' (SEQ ID NO: 4); The primer pair for specifically amplifying the mutant ko1-22 consists of forward primer F-2 and reverse primer R-2. Forward primer F-2: 5'-GCTTTAGGCGAGGATGTGAGTTC-3' (SEQ ID NO: 5) Reverse primer R-2: 5'-CGCGTTCAAGTACGGTAACTCTG-3' (SEQ ID NO: 6); The primer pairs mentioned above were synthesized by Nanjing Qingke Biotechnology Co., Ltd.

[0044] On the one hand, the above primer pair composition can be used for the detection of the photoperiod-temperature-sensitive male sterility gene in maize in Example 1. The genomic DNA of the maize to be tested was amplified by PCR using the above primer pair composition. The total volume of the PCR system was 25 μL, including 1 μL each of forward and reverse primers (10 μmol / L), 1 μL of genomic DNA, 12.5 μL of 2× Flash HS PCR Master Mix enzyme, and ddH2O to a final volume of 25 μL. The PCR reaction program was: 94℃ pre-denaturation for 1 min; 98℃ denaturation for 5 s, 58℃ annealing for 5 s, 72℃ extension for 10 s, for a total of 35 cycles; 72℃ over-extension for 2 min; and storage at 16℃. After sequencing the PCR amplification products, the PCR amplification products were then separated by 1% agarose gel electrophoresis. PCR products matching the target fragment size were sent to the company for Sanger sequencing. The sequencing peak diagram was submitted to SnapGene software for analysis, and the results are as follows: Figure 3 As shown, the wild-type and mutant plants are inconsistent at the mutation sites. This indicates that the marker can effectively distinguish between wild-type and mutant genes.

[0045] Taking the ko1-4 mutant mutation site as an example, the genotype detection method is as follows: (1) Using the genomic DNA of the maize material to be tested as a template, and using the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 above as primers, PCR amplification was performed; (2) The PCR products obtained in step (1) were subjected to Sanger sequencing, and then the peak diagram was interpreted using SnapGene software. If the mutation site is base G, the maize material to be tested is wild genotype; if the mutation site is base A, the maize material to be tested is a homozygous mutant genotype and photoperiod-sensitive male sterile material; if the mutation site is G / A, the maize material to be tested is a heterozygous genotype and male fertility is consistent with wild type material.

[0046] On the other hand, the combination of the above primer pairs can also be used to prepare a kit containing the above two primer pairs, which can detect the photoperiod-temperature-sensitive male sterility gene in maize.

[0047] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A photoperiod-temperature-sensitive male sterility gene mutant in maize, characterized in that, The mutant was obtained by a single-base mutation in the maize photoperiod-sensitive male sterility gene KO1. The KO1 gene is the Zm00001d046344 gene in the RefGen_v4 version of the inbred line B73 in the MaizeGDB database, and its DNA sequence is shown in SEQ ID NO:

1. The mutant is mutant ko1-4, mutant ko1-19, or mutant ko1-22. The mutant ko1-4 was obtained by changing the base from G to A at position 6329 of the KO1 gene DNA sequence starting from the start codon; The mutant ko1-19 was obtained by changing the base from G to A at position 5821 of the KO1 gene DNA sequence, starting from the start codon. The mutant ko1-22 was obtained by changing the base from G to A at position 5659 of the KO1 gene DNA sequence, starting from the start codon.

2. A primer pair composition for detecting a maize photoperiod-temperature-sensitive male sterility gene mutant as described in claim 1, characterized in that, The primer pair composition includes: a primer pair for the specific amplification mutant ko1-4, a primer pair for the specific amplification mutant ko1-19, or a primer pair for the specific amplification mutant ko1-22; The primer pair for the specific amplification mutant ko1-4 or the primer pair for the specific amplification mutant ko1-19 consists of a forward primer F-1 and a reverse primer R-1. The nucleotide sequence of the forward primer F-1 is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer R-1 is shown in SEQ ID NO:

4. The primer pair for specifically amplifying the mutant ko1-22 consists of a forward primer F-2 and a reverse primer R-2. The nucleotide sequence of the forward primer F-2 is shown in SEQ ID NO: 5, and the nucleotide sequence of the reverse primer R-2 is shown in SEQ ID NO:

6.

3. The use of a primer pair composition for detecting maize photoperiod-temperature-sensitive male sterility gene mutants as described in claim 2 in the preparation of a kit for detecting maize photoperiod-temperature-sensitive male sterility gene mutants.

4. A kit comprising the primer pair as described in claim 2 for identifying mutant ko1-4, mutant ko1-19, or mutant ko1-22.

5. The application of the photoperiod-temperature-sensitive male sterility gene mutant of maize as described in claim 1 in the breeding of dual-purpose male sterile maize lines.

6. The application of the photoperiod-temperature-sensitive male sterility gene mutant of maize according to claim 5 in the breeding of dual-purpose male sterile lines of maize, characterized in that: The dual-purpose male-sterile maize line is a maize material whose male fertility can switch between male sterility and fertility under different growth environments.

7. The application of the photoperiod-temperature-sensitive male sterility gene mutant of maize according to claim 5 in the breeding of dual-purpose male sterile lines of maize, characterized in that, The cultivation method includes the following steps: crossing a donor parent carrying a homozygous mutant ko1-4, ko1-19, or ko1-22 with a recipient inbred line to obtain the F1 generation; using the F1 generation as the maternal parent, backcrossing with the recipient inbred line for 3 to 5 generations, performing PCR amplification and sequencing on the composition using the primers described in claim 2 for each generation, identifying the genotype of the offspring plants based on the G / A base ratio at the mutation site, and selecting heterozygous plants carrying the target mutant gene and with a genetic background similar to the recipient parent for the next round of backcrossing; after the backcrossing is completed, obtaining a homozygous mutant dual-purpose sterile line with agronomic traits consistent with the recipient inbred line through self-pollination and molecular marker screening.

8. A method for detecting photoperiod-temperature-sensitive male sterility gene mutants in maize, characterized in that: Using the primer pair composition as described in claim 2, PCR amplification was performed on the genomic DNA of the maize to be tested. After sequencing the PCR amplification product, the genotype of the photoperiod-sensitive male sterility gene carried by the maize to be tested was determined based on the bases at the mutation site.

9. The method for detecting photoperiod-temperature-sensitive male sterility gene mutants in maize according to claim 8, characterized in that: The mutation site is selected from one of the following: The DNA sequence of the maize KO1 gene Zm00001d046344 has a mutation site at position 6329 from the start codon. The mutation site has a base G, indicating a wild-type genotype, and a mutation site has a base A, indicating a mutant genotype. The DNA sequence of the maize KO1 gene Zm00001d046344 has a mutation site at position 5821 from the start codon. The mutation site has a base G, indicating a wild-type genotype, and a mutation site has a base A, indicating a mutant genotype. The DNA sequence of the maize KO1 gene Zm00001d046344 has a mutation site at base 5659 from the start codon. The mutation site has a base G, indicating a wild-type genotype, and a mutation site has a base A, indicating a mutant genotype.

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