Development of functional markers for ems17, a recessive cytoplasmic male sterile mutant in maize and its application

Abstract

This invention relates to a recessive nuclear male sterility mutant of maize. ems17 This invention relates to gene mapping, molecular marker development, and breeding applications of a recessive nuclear male-sterile mutant obtained through EMS mutagenesis of the maize inbred line Zheng 58. ems17 Using the material, a map-based cloning strategy was employed to obtain... EMs17 This gene encodes a GDSL lipase. Utilizing... EMs17 An Indel functional marker was designed at the mutation site in the intron to specifically detect maize mutants. ems17 and the mutant genes in maize male-sterile materials bred from them. ems17 And can distinguish between wild type at the same time. EMs17 Genes and mutants ems17 Genes. The functional markers designed using this invention can accurately detect and label maize nuclear male sterility genes. ems17 Or fertile genes EMs17 The alleles can be used to identify male-sterile lines and screen target plants, and have important application value in the breeding of male-sterile maize lines, sterile hybrid seed production and molecular marker-assisted selection.

Description

Technical Field

[0001] Generally, this invention belongs to the fields of crop molecular breeding, molecular biology, and genetic engineering. Specifically, it relates to a functional marker for the maize recessive nuclear male sterility gene ems17, a method for developing the functional marker, and its application. Background Technology

[0002] Male sterility (MS) in plants refers to the phenomenon in which male organs develop abnormally and are unable to produce functional male gametes (pollen), while female organs develop normally, can accept normal male gametes for fertilization and fruit formation, and can pass on this sterility to offspring.

[0003] Maize is an important food crop, and its male-sterile materials are of great value for research on the development mechanism of male flowers, genetic breeding applications, and heterosis research and applications. Maize male sterility can be divided into three categories according to the difference in the mode of sterility inheritance: cytoplasmic male sterility, nuclear male sterility, and nuclear-cytoplasmic interaction male sterility. Nuclear male sterility can be further divided into dominant nuclear male sterility and recessive nuclear male sterility, with the latter being the predominant type. Due to maternal inheritance in the cytoplasm, the F1 generation of cytoplasmic male sterility genes cannot self-pollinate and thus cannot be utilized in breeding and production. Maintaining and propagating nuclear male sterile lines using conventional hybridization breeding techniques is difficult, making them unsuitable for effective breeding and production. Nuclear-cytoplasmic interaction male sterility genes can be used in breeding both theoretically and practically; however, the widespread use of these genes can lead to cytoplasmic homogenization in hybrid varieties, making them susceptible to infection by specific pathogen races and posing significant risks to hybrid maize production. Therefore, most of the parent lines for maize hybrid breeding at home and abroad are fertile inbred lines. When producing hybrid seeds, the female parent needs to be manually or mechanically demasked, which greatly increases the cost of seed production. At the same time, it is difficult to guarantee the purity of the hybrid seeds.

[0004] With the rapid development of modern biotechnology, it is hoped that recessive nuclear male sterility genes can be effectively utilized by combining crop molecular design techniques with conventional breeding methods. To enable the breeding application of nuclear male sterility genes, it is essential to identify marker traits for early diagnosis of sterility in nuclear male sterile offspring, thereby distinguishing nuclear male sterile lines as early as possible. Therefore, since the discovery of recessive nuclear male sterile materials in maize, researchers have explored various marker traits for nuclear male sterility genes using traditional breeding techniques. For example, they have developed grain color marker systems, yellow-green seedling linkage marker systems, and multi-filament linkage marker systems based on the close linkage between marker traits and sterility. However, due to problems such as incomplete linkage between marker traits and sterility, difficulties in marker trait identification, and delayed identification time, these methods and attempts have not been widely applied in maize production. Molecular markers are genetic markers based on nucleotide sequence variations within the genetic material of individuals, and are a direct reflection of genetic polymorphism at the DNA level. In a broad sense, molecular markers refer to heritable and detectable DNA sequences or proteins. In a narrow sense, molecular markers refer to specific DNA fragments that reflect certain differences in the genomes of individual organisms or populations. With the development of molecular marker technology, obtaining molecular markers linked to important traits in maize or functional co-segregating molecular markers through localized cloning is of great significance for maize genotyping, selection of genetic background for varieties, screening of target plants, genetic improvement and purity identification of varieties, and cloning genes controlling male organ differentiation. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a set of molecular markers that can accurately detect and label the recessive nuclear male sterility gene ems17 in maize. The provided molecular markers are designed based on the gene mutation sites of sterility mutants and belong to functional co-segregating molecular markers. They co-segregate with the sterility gene ems17 and can be used for identification of fertility alleles in plants, screening of target individual plants in molecular marker-assisted breeding, and identification of seed purity.

[0006] One of the objectives of this invention is to provide a completely male-sterile material obtained due to a mutation in the fertility-related gene ZmEMs17.

[0007] The second objective of this invention is to provide the gene mutation site for this mutant material. Specifically, the gene mutation is caused by a 14bp sequence deletion starting at the +1503 site.

[0008] The third objective of this invention is to provide a method for developing functional molecular markers of ems17 based on the aforementioned ems17 mutation sites.

[0009] The fourth objective of this invention is to provide a set of functional molecular markers developed based on the above-mentioned mutation sites, including but not limited to the ems17-Indel functional marker, which can simultaneously detect the mutant allele ems17 and the wild-type allele EMs17.

[0010] Furthermore, the developed molecular marker ems17-Indel and other similar functional co-segregating markers include, but are not limited to, the following two primer sequences:

[0011] ems17-Indel-F:CAGCGACTACCGTGATGGA

[0012] ems17-Indel-R:ACCCTGCTCCTGTGCTTAGG

[0013] The forward primer sequence ems17-Indel-F was designed based on the upstream sequence of the 14bp deletion sequence of the ems17 gene intron, and the forward primer sequence ems17-Indel-R was designed based on the downstream sequence of the deletion sequence. Using these primers, a 123bp fragment was amplified in wild-type DNA, while a 109bp fragment was amplified in mutant DNA. The wild-type DNA and the sterile mutant DNA could be clearly distinguished using polyacrylamide gel electrophoresis.

[0014] The fifth objective of this invention is to provide a method for developing and selecting maize male-sterile materials with different genetic backgrounds using the aforementioned mutant materials. Specifically, materials carrying the aforementioned mutant gene ems17 are used as the female parent, and inbred lines with different superior genetic backgrounds are used as the male parent. The hybrid offspring are backcrossed with the male parent inbred lines for multiple generations. Before each backcross, markers are used to select individual plants carrying the ems17 gene to obtain maize ems17 male-sterile materials with different genetic backgrounds, thereby expanding the scope of heterosis utilization. Attached Figure Description

[0015] Figure 1 Comparative images of the tassels and florets (anthers) phenotypes of wild-type maize EMs17 (left) and EMs17 mutant (right), as well as comparative images of pollen iodine-potassium iodide staining.

[0016] Figure 2 The segregation ratio of fertile to sterile plants in the F2 and F2:3 families of the maize ems17 mutant.

[0017] Figure 3 Gene mapping and candidate gene analysis were performed for the maize ems17 mutant. ems17 was located within a 517.867 kb region between Chr4 189.054 Mb and 189.572 Mb, with 18 candidate genes identified.

[0018] Figure 4 The expression pattern of candidate gene Zm00001d052403 at different stages of maize anther development was analyzed. A significant expression peak was observed in Zm00001d052403 during the S7-S8b stage of anther development, suggesting it is a potential candidate gene.

[0019] Figure 5 Sequencing analysis of the protein encoded by the Zm00001d052403 gene revealed a mutation at bases 1075-1077 in the CDS region to TAG, and a stop codon mutation at amino acid 357, prematurely halting translation. This resulted in a 25-amino acid deletion in the protein encoded by the ems17 mutant Zm00001d052403 gene.

[0020] Figure 6 Structural comparison analysis of the wild-type, mutant, and ms30-6028 genes of Zm00001d052403 was performed. Zm00001d052403 was named EMs17, which encodes a GDSL lipase protein (GELP). ems17 is a novel allelic mutation of the ZmMs30 mutant ms30-6028.

[0021] Figure 7 The development and population validation of Indel functional markers for the ZmEMs17 gene were carried out. A. Differences in intron sequences between fertile and sterile DNA of ems17; B. ems17-indel marker polymorphism test; C. ems17-indel marker linkage test; D. Polyacrylamide gel electrophoresis of partial population tests of ems17-indel markers. The Indel markers of the ZmEMs17 gene are fully linked to the ems17 mutant population material and can be used as specific functional markers for marker-assisted breeding.

[0022] Figure 8 A schematic diagram illustrating the technical route for creating a new genetic background maize ems17 nuclear male-sterile line through backcrossing using molecular marker-assisted selection. Detailed Implementation

[0023] The following embodiments are used to illustrate the present invention, but do not limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. Unless otherwise specified, the synthesis and sequencing of primers and genes used in the embodiments were performed by Sangon Biotech (Shanghai) Co., Ltd. Other biochemical reagents, unless otherwise specified, are conventional commercially available reagents, and the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0024] Example 1: Obtaining male-sterile mutant maize material by EMS chemical mutagenesis

[0025] The test material of this invention is a male-sterile mutant material obtained from the EMS mutagenesis mutant library of the normal fertile maize inbred line Zheng 58, exhibiting complete pollen-free male sterility, such as... Figure 1 As shown, the specific manifestations are: the anthers become smaller and whiter, and the pollen cannot be stained by iodine-potassium iodide. This sterile line is named ems17.

[0026] Example 2: Construction and genetic analysis of segregating populations of ems17 mutants

[0027] Using the ems17 sterile mutant as the female parent and the conventional inbred line B73 as the male parent, a segregating F2 generation was constructed through hybridization. In the winter of 2022, F2 generation seeds were planted at the experimental base in Sanya City, Hainan Province. The F2 generation plants were bagged and self-pollinated to obtain F2:3 family seeds. Leaves from the F2 segregating population were used for preliminary gene mapping of the ems17 mutant, while leaves from the F2:3 family plants obtained through self-pollination were used for fine mapping of the ems17 mutant gene.

[0028] To conduct genetic analysis of the sterility gene in the ems17 mutant, B73 was used as the male parent and the ems17 sterile mutant as the female parent in a cross. The resulting F1 generation seeds were planted and their phenotypes were observed in the field at maturity. The results showed that all F1 plants exhibited the same normal male fertility trait as the male parent B73, indicating that the ems17 mutant gene is recessive. After self-pollination of the F1 plants, F2 segregating population seeds were produced. These F2 segregating population seeds were sown and, until flowering and pollination, the tassel fertility phenotype of 446 maize plants was observed. The results are shown below. Figure 2 Of the 342 plants exhibiting fertility, 104 exhibited sterility, resulting in a fertility segregation ratio of 3.3:1. Chi-square test confirmed that the fertility ratio of the F2 generation segregating population conformed to Mendel's law of inheritance (3:1). Further genetic analysis of the sterility gene in the ems17 mutant was conducted. F2:3 family seeds were obtained by self-pollination of fertile F2 plants. In the following season, from the sowing of these F2:3 family seeds until flowering and pollination, the tassel fertility phenotype of 1056 maize plants was observed. 801 plants exhibited fertility, and 255 exhibited sterility, resulting in a fertility segregation ratio of 3.14:1. Chi-square test confirmed that the fertility ratio of the F2:3 family segregating population conformed to Mendel's law of inheritance (3:1). Therefore, the sterility gene in the ems17 mutant was determined to be a recessive nuclear sterility gene controlled by a single gene.

[0029] Example 3: Fine mapping of ems17 mutations and expression analysis of candidate genes

[0030] The male sterility gene was initially mapped using a segregating F2 generation of B73×ems17. A total of 129 maize plants (including 25 fertile plants and 104 sterile plants) were sampled. SSR linkage marker analysis initially mapped the target gene to markers M1-M7 (Chr4 185Mb-198Mb), with a physical distance of 13Mb between the initial mapping intervals. To narrow down the gene mapping interval and more precisely identify candidate genes, a segregating F2:3 generation family was further planted. A total of 1056 maize plants were sampled, including 801 fertile plants and 255 sterile plants. The population was expanded using 300 maize plant DNA samples (255 sterile plant DNA samples and 45 fertile plant DNA samples) for fine mapping of the male sterility gene ems17. SSR molecular markers with polymorphism and close linkage to the target gene, screened in the fine mapping experiment, were used to screen single plants that exchanged DNA between all sterile plants and some fertile plants in the F2:3 population. Ultimately, the target gene was located within a 517.867 kb interval between M15 and M14 (Chr4-189.054 Mb-189.572 Mb), which included 18 candidate genes. Figure 3 The proteins encoded by 18 candidate genes and their expression patterns in different tissues were analyzed using the National Center for Biotechnology Information (NCBI) database. Figure 4 The results showed that among the 18 candidate genes, Zm00001d052412 and Zm00001d052413 were not expressed in any maize tissues, Zm00001d052415 was specifically expressed in anthers but not in other tissues, Zm00001d052403 was highly expressed in anthers, and the remaining 14 genes were expressed in all maize tissues, indicating constitutive expression. Zm00001d052403 encodes a GDSL esterase / lipase protein; this protein family has been reported to regulate maize fertility and could be considered an important candidate gene.

[0031] Example 4: Sequencing analysis of key candidate genes

[0032] To determine whether there are sequence differences in the key candidate gene Zm00001d052403 between the male-sterile mutant ems17 and the wild type, genomic cloning and sequencing analyses were performed on both candidate genes. First, PCR amplification experiments were conducted using genomic DNA from the male-sterile mutant ems17 and the wild-type B73 inbred line. The alignment results of the DNA and protein sequences of the CDS region of the Zm00001d052403 gene are shown below. Figure 5This indicates that a large base mutation occurs at position 1071 bp in the CDS gene sequence of the ems17 mutant, with a TAG mutation at positions 1075-1077. During transcription and translation, this mutation becomes a stop codon, prematurely halting translation and causing a 25-amino acid deletion in the protein encoded by the ems17 mutant Zm00001d052403 gene, potentially affecting the structure and function of the encoded protein. Comparison of the candidate gene wild-type, mutant, and ms30-6028 gene structures reveals that ems17 is a novel allelic variant of ms30-6028. Figure 6 Therefore, it is preliminarily speculated that Zm00001d052403 is the sterility gene of the ems17 mutant, and the premature termination of translation of this gene in the ems17 mutant may be an important reason for the male sterility phenotype.

[0033] Example 5: Indel functional marker design and population validation of the ZmEMS17 gene

[0034] In maize breeding, In / Del markers are widely used in genetic diversity analysis, variety identification, trait association studies, and marker-assisted selection (MAS). To verify whether the male sterility gene in the ems17 mutant is Zm00001d052403, sequencing results of Zm00001d052403 revealed a 14bp deletion at position 465bp in the second intron of the ems17 male sterility mutant sequence compared to the wild-type sequence. Figure 7 Subsequently, indel primers, named ems17-indel, were designed at both ends of the deleted sequence using Primer Premier 5 software. Primer sequence information is shown in the sequence listing. This indel marker exhibited polymorphism and linkage in PCR amplification experiments of fertile and sterile DNA. Figure 7 The DNA fragment amplified to 123 bp in wild-type DNA and to 109 bp in mutant DNA. Polyacrylamide gel electrophoresis clearly distinguished the wild-type DNA from the sterile mutant DNA. F was used for fine localization. 2：3 A total of 300 maize accessions (255 sterile plants and 45 fertile plants) from the segregating family population were amplified by PCR using ems17-indel labeled primers and analyzed by PAGE electrophoresis. PAGE gel results for some large populations are shown below. Figure 7The results showed that no single plant exchanged the Zm00001d052403 gene between fertile and sterile plants, indicating that the Zm00001d052403 gene was completely linked to the ems17 mutant population, further verifying that Zm00001d052403 is the sterility gene of the ems17 mutant. Therefore, the sterility gene of ems17 was named ZmEMs17. Furthermore, this Indel marker has the potential for screening ems17 mutants and for molecularly assisted breeding with sterilization. Figure 8 ).

[0035] The method for extracting DNA from corn leaves is as follows: 1) Cut an appropriate amount of leaves and chop them into small pieces. Place them into 2.0ml centrifuge tubes that have been pre-labeled with numbers, and add a steel ball. 2) Arrange the centrifuge tubes containing the leaves and steel ball in order on a centrifuge tube rack (8×5) for the sampler, and immerse the entire tube in a container filled with liquid nitrogen for 1-2 minutes (Note: the liquid nitrogen should just cover the centrifuge tube rack slightly). 3) Place the frozen centrifuge tube rack into the slot of the sampler (Thmorgan Cell Killer CK-1000), tighten it, close the cap, and sample at 1200 rpm for 20 seconds. 4) Use a magnet to remove the steel ball from the centrifuge tube. 5) Add 700μL LCTAB extraction buffer (preheated to 65℃), and incubate in a 65℃ water bath for 30 minutes, inverting the tube 1-2 times during the incubation period. 6) Add 700 μL of chloroform:isoamyl alcohol (24:1) extraction buffer, tighten the cap, and mix by inverting the tube. Be careful not to erase the label (Note: Chloroform is a corrosive reagent; wear disposable PE gloves and handle in a fume hood). 7) Centrifuge at 12000 rpm for 5 min until clear phase separation is achieved. Transfer 400 μL of the supernatant to a new 1.5 mL microcentrifuge tube (pre-filled with 800 μL of pre-chilled anhydrous ethanol), discard the pipette tip, tighten the cap, label and check for accuracy, and mix by inverting the tube. Place in a -20℃ refrigerator for 30 min. 8) Centrifuge at 12000 rpm for 10 min until the precipitate adheres to the bottom of the centrifuge tube, and discard the supernatant. 9) Wash the precipitate twice with 70% ethanol, and place the 1.5 mL microcentrifuge tube upside down on a piece of paper laid flat on the table to air dry. 10) Add 100-200 μL of 1×TE buffer or ddH2O to dissolve the precipitate. 11) Store the sample in a -20℃ refrigerator for later use.

[0036] The obtained DNA was amplified by PCR using the designed primers. The method and procedure are as follows: 1) First, turn on the water supply switch of the ice maker, then turn on the power switch of the ice maker to make ice. 2) Take the following reagents out of the -20℃ freezer to thaw: PCR buffer, dNTP solution, forward and reverse primer solutions, and template DNA (Note: Once a reagent is completely thawed, place it on ice. Commonly used ddH2O, primer working solution, template DNA, and a small amount of PCR buffer and dNTP solution can be placed in a 4℃ freezer). 3) After all reagents have thawed, centrifuge at 8000 rpm for a few seconds and place back on ice for later use. 4) Prepare the PCR reaction mixture by adding each reagent in the order shown in the table below (the reaction volume is 10 μL). Table 1 below lists the mixture formulas for 1 reaction (1R×), 10 reactions (10R×), 50 reactions (50R×), and 100 reactions (100R×). After preparation, return all reagents to a 4℃ or -20℃ freezer (Note: Taq polymerase requires careful handling; remove it from the -20℃ freezer just before use, place it on ice during use, and immediately return it to the -20℃ freezer after use). 5) Mix well and centrifuge at 8000 rpm for a few seconds. 6) Aliquot the mixture into 200μL PCR reaction tubes, add 1μL template DNA, and label them (Note: If the hot cap function is not used, to prevent the sample from drying out, add approximately 20μL of paraffin oil or cap the PCR tubes). 7) Insert the PCR reaction plate (tube) into the 200μL well of the PCR amplification instrument, close the hot cap, and tighten it. 8) Turn on the PCR amplification instrument, select the preset reaction program (as shown in Table 2), and start amplification. 9) After amplification, exit the reaction program, return to the main menu, and turn off the power switch. Open the hot cap, remove the PCR reaction tubes, place them on the PCR tube rack, and store them in a 4℃ freezer for later use.

[0037] Table 1. PCR reaction system

[0038]

[0039]

[0040] Table 2. PCR amplification conditions

[0041]

[0042] Note: The annealing temperature varies depending on the primer, and is generally between 56℃ and 60℃.

[0043] In summary, the molecular marker ems17-indel developed in this invention is a functional molecular marker for the maize nuclear male sterility gene ems17. Combined with the molecular marker of the wild-type EMs17 gene, it allows for rapid detection of EMs17 allele status. During backcrossing and conversion, marker-assisted selection can directly detect lines carrying the ems17 gene at the seedling stage, rapidly screening for target individuals to accelerate the backcrossing and conversion process. This has significant application value in marker-assisted selection for hybrid sterilization seed production. Furthermore, the developed molecular marker can also be used to identify the purity of sterile parents in hybrids.

[0044] Related literature

[0045] 1. Beadle, GW (1932) Genes in maize for pollen sterility. Genetics, 17: 413-4312, Albertsen, MC and Phillips, RL (1981) Developmental cytology of 13 genetic male sterile lociin maize. Can J Genet Cytol, 23: 195-208

[0046] 3. Wu Suowei, Fang Caichen, Deng Lianwu, Wan Xiangyuan (2012). Research progress on recessive nuclear male sterility gene in maize and its application in breeding. Molecular Plant Breeding (Online Edition), 10: 1001-1011.

[0047] 4. Wu Suowei, Wan Xiangyuan (2018) Establishing a technical system for male-sterile hybrid breeding and seed production of major crops using biotechnology. China Biotechnology Journal, 38(1): 78-87

[0048] 5. Liu Shuangshuang, Wu Suowei, Rao Liqun, Wan Xiangyuan (2018). Study and application analysis of molecular mechanism of male sterility in maize kernels. China Biotechnology Journal, 38(1): 100-107.

[0049] 6. Wan Xiangyuan, Xie Ke, Wu Suowei, An Xueli, Li Jinping, Zhang Danfeng, Liu Shensi, Xiao Zhonghua, A method for maintaining and propagating maize male-sterile lines based on the Ms30 gene, February 22, 2017, Chinese Invention Patent, ZL201510300778.9

[0050] 7. Wan Xiangyuan, Wu Suowei, Zhou Yan, Xie Ke, Li Jinping, DNA sequence of EMs17, a gene regulating postmeiotic development of maize pollen, and its encoded protein, April 12, 2017, Chinese Invention Patent, ZL201410703778.

Claims

1. A maize nuclear male sterility mutant ems17 The functional marker ems17-Indel is characterized by, The functional markers include, but are not limited to, the first primer ems17-Indel-F and the second primer ems17-Indel-R: ems17-Indel-F:CAGCGACTACCGTGATGGA ems17-Indel-R:ACCCTGCTCCTGTGCTTAGG.

2. The functional marker described in claim 1 is based on a maize nuclear male sterility mutant. ems17 mutated genes ems17 The design of mutation sites is a functional molecular marker, wherein the molecular marker described in claim 1 is capable of simultaneously detecting... EMs17 and ems17 Alleles.

3. The nuclear male sterile mutant according to claim 2 ems17 Its characteristics are, Its mutated gene ems17 Located on chromosome 4 of maize, corresponding to the wild-type fertile gene EMs17 It affects the developmental progress of pollen microspores, thereby mediating the developmental ability of male flowers, and its mutations lead to male flower sterility.

4. Similar to claims 1, 2, and 3, the present invention... ems17 Other gene mutation site designs related to e ms17 Functional markers and development of gene cosegregation ms17 Methods for functional labeling.

5. The maize male development-related genes as described in claims 1, 2, 3, and 4 Ems17 and mutated genes ems17 Molecular markers were used to detect wild-type cells. EMs17 and mutated genes ems17 Applications in [the field].

6. The maize male development-related mutant gene as described in claim 1 ems17 Molecular markers and the maize nuclear male sterility gene e as described in claim 5 ms17 Application in breeding and seed production of maize sterile lines.

7. The maize male development-related mutant gene as described in claim 1 ems17 A method for applying molecular markers to breed new maize male-sterile lines with novel genetic backgrounds, characterized by, With maize nuclear male sterility gene ems17 Using maize as the female parent and other maize inbred lines with superior traits as the male parent, a new maize nuclear male-sterile line with a new genetic background is obtained through hybridization and multiple rounds of crosses.

8. The maize male development-related mutant gene as described in claim 1 ems17 Molecular markers in rapid detection of maize nuclear male sterility genes ems17 Applications of alleles.

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