A molecular marker related to the starch content of corn kernels and use thereof

Abstract

The application discloses a molecular marker related to corn kernel starch content and application thereof, and relates to the technical field of biology.The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3.A key gene ZmEnd1 for regulating corn starch content is successfully mined for the first time, and two key SNP sites, namely SNP-4-245033385 and SNP-4-245033215, located in the promoter region of the ZmEnd1 gene are found, and the two sites are significantly related to the corn kernel starch content.Based on the above research results, the application further develops a molecular marker closely related to the corn kernel starch content.Through the use of the molecular marker, researchers can quickly and accurately detect the genetic characteristics of the starch content in the corn kernel, thereby providing strong technical support for the breeding of corn varieties with high starch content.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a molecular marker related to the starch content of corn kernels and its application. Background Technology

[0002] Maize (Zea mays L.) is one of the most important food crops and an essential source of feed and industrial raw materials. The kernel, as the most important harvested organ of the maize plant, typically contains over 70% starch by dry weight, making it the most important form of energy storage. In cereals such as maize and rice, starch content not only determines kernel weight and volume but also affects processing performance and the quality of the final product. Therefore, identifying genes regulating kernel starch content and deeply analyzing the molecular mechanisms of starch synthesis and accumulation are of significant theoretical and practical importance for improving maize yield and kernel quality. However, traditional breeding methods rely on phenotypic selection, which is limited by the time-consuming and costly nature of kernel starch content detection and the interference of environmental factors, making it difficult to achieve efficient and precise genetic improvement.

[0003] With the rapid development of molecular biology techniques, molecular marker technology can significantly improve breeding efficiency. Molecular marker technology is a tool for revealing genetic differences in individuals or populations at the molecular level, based on sequence differences at specific DNA loci among individuals. Closely linked to target traits, it enables rapid, efficient, and precise selection of target traits, shortening the breeding cycle. Genome-wide association studies (GWAS) have been widely used in the genetic basis research of complex quantitative traits in plants. Therefore, identifying genes regulating starch content in maize kernels and developing their molecular markers is of great significance for maize genetic improvement. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular marker related to starch content in maize kernels and its application, thereby addressing the problems existing in the prior art. Using this molecular marker, researchers can quickly and accurately detect the genetic characteristics of starch content in maize kernels, thus providing strong technical support for the breeding of maize varieties with high starch content.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a molecular marker related to starch content in maize kernels, the nucleotide sequence of which is shown in SEQ ID NO.3. The molecular marker includes two SNP sites, SNP-4-245033215 and SNP-4-245033385. SNP-4-245033215 is located at the 1751st base of the nucleotide sequence, and SNP-4-245033385 is located at the 1921st base of the nucleotide sequence.

[0007] The present invention also provides a dCAPS primer set, comprising an upstream primer dCAPS-F with a nucleotide sequence as shown in SEQ ID NO.26, an upstream primer dCAPS-F-mutant with a nucleotide sequence as shown in SEQ ID NO.27, and a downstream primer dCAPS-R with a nucleotide sequence as shown in SEQ ID NO.28.

[0008] This invention also provides the application of the above-mentioned dCAPS primer set in the preparation of detection products for identifying the starch content trait of corn kernels.

[0009] Furthermore, the testing product is a reagent kit.

[0010] The present invention also provides a detection product for identifying the starch content trait of corn kernels, comprising the above-mentioned dCAPS primer set.

[0011] Furthermore, the testing product is a reagent kit.

[0012] The present invention also provides the application of the above-mentioned molecular markers in identifying the starch content trait of corn kernels.

[0013] The present invention also provides the application of the above-mentioned dCAPS primer set in identifying the starch content trait of corn kernels.

[0014] The present invention also provides the application of the above-mentioned detection product in identifying the starch content trait of corn kernels.

[0015] This invention also provides a method for identifying the starch content trait of corn kernels, comprising the following steps:

[0016] Genomic DNA was extracted from the sample to be tested;

[0017] Using the genomic DNA as a template, PCR amplification was performed using the above-mentioned dCAPS primer set to obtain the haplotype of the sample to be tested, and the starch content trait of corn kernels was determined: the starch content of corn kernels of haplotype AA was higher than that of haplotype GG.

[0018] The present invention discloses the following technical effects:

[0019] This invention marks the first successful discovery of a key gene regulating corn starch content—ZmEnd1—providing crucial clues for in-depth research into the starch metabolism mechanism of corn kernels. Furthermore, this invention also identified two key SNP sites located in the promoter region of the ZmEnd1 gene, namely SNP-4-245033385 and SNP-4-245033215, which are significantly associated with corn kernel starch content.

[0020] Based on the above research findings, this invention further develops a molecular marker closely related to the starch content of maize kernels. By utilizing this molecular marker, researchers can rapidly and accurately detect the genetic characteristics of starch content in maize kernels, thus providing strong technical support for the breeding of maize varieties with high starch content. This technological breakthrough not only improves the efficiency of maize genetic improvement but also lays a solid foundation for the targeted improvement of maize quality, possessing significant scientific value and broad application prospects. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The results of detecting significant SNPs controlling grain starch content in four environments and BLUP are shown in the figure. Among them, (a) is the Manhattan plot of SNPs significantly associated with grain starch content, and the positions of the red boxes are the sites located in the four environments and BLUP; (b) is the QQ plot of SNPs significantly associated with grain starch content; 2020XSBN, 2021CZ, 2021YA, and 2021XSBN represent Xishuangbanna Dai Autonomous Prefecture, Yunnan Province in 2020, Chongzhou City, Sichuan Province in 2021, Ya'an City, Sichuan Province in 2021, and Xishuangbanna Dai Autonomous Prefecture, Yunnan Province in 2021, respectively; (c) is the expression pattern diagram of candidate genes in different tissues.

[0023] Figure 2 The figure shows the detection results of the difference in transcriptional activity caused by the ZmEnd1 promoter variation. Among them, (a) is the result of the ZmEnd1 candidate gene association analysis; (b)-(d) are the phenotypic values ​​of grain starch content of different haplotypes in Chongzhou City (CZ) and Ya'an City (YA) of Sichuan Province in 2021, and the best linear unbiased prediction (BLUP), respectively; *** represents the significance of the difference at the P<0.001 level; ** represents the significance of the difference at the P<0.01 level; (e) is the result of fluorescence imaging of ZmEnd1 promoter activity; (f) is the result of quantitative comparison of Hap1 and Hap2 promoter activities; *** represents the significance of the difference at the P<0.001 level.

[0024] Figure 3The diagram shows the development of dCAPS markers; (a) is a gel electrophoresis diagram of dCAPS identifying different haplotypes; (b) shows the location of the restriction enzyme cleavage site, with red bases indicating the location of the marker, black sequences indicating the sequence of the promoter marker, blue sequences indicating F primers with mutation sites, and bolded bases indicating mutated bases; (c) shows the NcoI restriction enzyme recognition site.

[0025] Figure 4 Figure 1 shows the results of ZmEnd1 expression pattern analysis. (a) shows the expression pattern analysis results of ZmEnd1 at different stages of embryo, endosperm, and grain in the B73 inbred line; (b) shows the in situ hybridization results of ZmEnd1 in grains 15 days after pollination of the B73 inbred line; (c) shows the expression of ZmEnd1 in different parts and at different developmental stages of grains; different letters represent significant differences; (d) shows the statistical graph of ZmEnd1 expression levels at different developmental stages in grains of different haplotypes; ***, **, and * represent significant differences of P<0.001, P<0.01, and P<0.05, respectively.

[0026] Figure 5 To detect the subcellular localization of the ZmEnd1 gene in tobacco leaves; (a) shows schematic diagrams of the pCAMBIA1305-35S-eGFP and pCAMBIA1305-35S-eGFP-ZmEnd1 vectors; (b) shows the subcellular localization of the ZmEnd1 gene in tobacco leaves; scale bar is 30 μm; (c) shows the fluorescence detection results of ZmEnd1 in chloroplast channels, scale bar = 30 μm;

[0027] Figure 6 The results of yield trait testing of maize inbred lines ZmEnd1 mutant and B73 are shown. Among them, (a) is a phenotypic trait comparison chart; (b)-(g) are statistical charts of ear length, 100-kernel weight, kernel length, kernel width, ear diameter and kernel thickness, respectively; ***, ** and * represent significant differences at the P<0.001, P<0.01 and P<0.05 levels, respectively, and ns indicates no significant difference.

[0028] Figure 7 This is a statistical chart of the content of corn kernels; where (a)-(d) are statistical charts of the content of UDP-glucose, starch, glucose-1-P and trehalose-6-phosphate, respectively.

[0029] Figure 8 The images show the scanning electron microscopy (SEM) results of maize kernels from the ZmEnd1 mutant and the B73 inbred line. (a) shows the SEM images of maize kernels at different growth stages. (b)-(d) are statistical graphs of starch grain diameters at 15 DAP, 20 DAP, and 25 DAP, respectively.

[0030] Figure 9Figure 1 shows the phenotypic identification results of heterologous expression of the ZmEnd1 gene in rice. Among them, (a) is a comparison of yield traits of heterologous expression of ZmEnd1 in rice; Nip, wild-type Nipponbare rice; (b) is the semi-quantitative analysis results of ZmEnd1 in transgenic grains; (c) is the result of the thousand-grain weight detection of ZmEnd1 overexpressing rice lines and wild-type Nipponbare rice; (d) is the result of the grain starch content detection of ZmEnd1 overexpressing rice lines and wild-type Nipponbare rice; (e) is the result of the grain glucose-1-phosphate content detection of ZmEnd1 overexpressing rice lines and wild-type Nipponbare rice; ***, ** and * represent significant differences of P<0.001, P<0.01 and P<0.05, respectively. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] The primer information involved in this invention is shown in Table 1.

[0037] Table 1 Primer Information

[0038]

[0039]

[0040] Example 1

[0041] I. Experimental Methods

[0042] 1. Using GWAS to identify genetic loci regulating starch content in maize kernels.

[0043] Linked populations were planted in Xishuangbanna Dai Autonomous Prefecture, Yunnan Province (22.02°N, 100.80°E) (2020XSBN), Chongzhou City, Sichuan Province (30.32°N, 103.38°E) (2021CZ), Ya'an City, Sichuan Province (9.59°N, 102.57°E) (2021YA), and Xishuangbanna Dai Autonomous Prefecture, Yunnan Province (22.02°N, 100.80°E) (2021XSBN), respectively. A completely randomized block design was used, with a row length of 3m, a row spacing of 0.7m, and 12 plants per row. Five ears of fruit with uniform growth were taken from each population. The kernels from the middle part of the ears were dried at 60℃ for 2 days. The starch content of the kernels was collected using a DA7250 wavepass near-infrared spectrometer, and the technical replication was performed three times. Based on the high-density genotypes containing 969,439 SNP loci already available from the inventors' research group, GWAS analysis was performed using the FarmCPU model in the R package rMVP in R studio. The significance threshold for GWAS was set to P = 0.05 / number of valid markers, i.e., P = 1E. -06 .

[0044] 2. Candidate gene association analysis was used to identify key genes and significant variation sites related to grain starch content.

[0045] The stable SNP (SNP-4-245041154) detected under GWAS and BLUP was compared. Eight candidate genes were identified within the LD (=50Kb) region of this SNP. Analysis of expression patterns in different tissues showed that Zm00001d054004 (named ZmEnd1) was significantly highly expressed in grain endosperm.

[0046] The nucleotide sequence of the ZmEnd1 gene is shown in SEQ ID NO.1.

[0047] Further candidate gene association analysis was used to verify the regulatory role of this gene on maize kernel starch content. Using DNA from 285 maize inbred lines in the associated population as templates, the upstream 2000 bp of the ZmEnd1 gene and the gene itself were amplified from the genome using PCR and sequenced. Sequencing results were assembled and aligned using the DNAMAN program (V version 5.2.2, LynnonBio-soft, Canada). DNASP v.5.0 was used to detect nucleotide polymorphisms, including SNPs and InDels. Based on the kernel starch content phenotypic data of the 285 inbred lines under four environments and BLUP, SNPs / InDels with a minimum allele frequency (MAF) ≥ 0.05 and a heterozygosity rate (HR) < 20% were selected. Association analysis was performed using the FarmCPU model in TASSEL 5.0 (significance threshold set to: P = 0.05 / number of effective markers). In this invention, variant sites (P<0.001) are considered to be significantly correlated with grain starch content. Haplotypes are classified based on significantly correlated SNPs and InDels. Linkage disequilibrium (LD) decay between adjacent markers is calculated using Haploview software. A t-test is used to analyze the significance of grain starch content among different haplotypes. Haplotypes with higher starch content are defined as superior haplotypes.

[0048] 3. Detection of promoter haplotype activity using dual-luciferase assay.

[0049] To detect the promoter activities of the two haplotypes, this invention amplified and sequenced the sequence 2000 bp upstream of the transcription start site in the material containing the haplotypes. Using Hind III (AAGCTT) and BamHI (GGATCC) as restriction enzyme sites, the two haplotypes of ZmEnd1 were constructed into the pGreenII-0800-LUC vector (P800) using homologous recombination, named P800-Hap1 (AA) and P800-Hap2 (GG). Primers for vector construction are detailed in Table 1, and the vector map is shown in [Table 1]. Figure 1 The constructed vectors P800-Hap1(AA) and P800-Hap2(GG) were then transformed into Agrobacterium GV3101-p19, respectively. The promoter activities represented by the two haplotypes were verified using a transient expression system in tobacco. The specific steps are as follows:

[0050] (1) Take 30 μL of competent cells transformed with P800-Hap1(AA) and P800-Hap2(GG) vectors and spread them on YEP medium containing resistance. Incubate at 28°C in the dark for two days.

[0051] (2) Pick single clones and put them into 1 mL of YEP liquid medium with the corresponding antibiotics (Kan and Rif added at a ratio of 1:1000) and culture them at 28°C and 200 rpm for 12 h.

[0052] (3) Use PCR technology to identify the bacterial culture and electrophoresis to determine whether the size of the inserted band is correct, that is, whether the vector has been successfully transformed into Agrobacterium competent cells.

[0053] (4) Transfer 100 μL of the positive bacterial suspension to 50 mL of YEP medium containing antibiotics (Kan and Rif at a ratio of 1:1000) and culture for 18 h. When the OD600 reaches 1.0, centrifuge at 4000 rpm for 10 min to collect the bacterial cells. Resuspend the bacterial cells in 10 mM MgCl2 (AS and MES added at a ratio of 1:1000) suspension and adjust the OD600 of the suspension to 0.8.

[0054] (5) Sow tobacco seeds in a culture box and culture them for about 40 days under conditions of 12h light / 10h darkness, 25℃ temperature, and 60%-70% humidity. Select tobacco plants with good growth and smooth leaves and inject the suspension from the back using a 1mL syringe. To ensure the accuracy of the experimental results, this invention injects the suspensions of the two haplotypes into symmetrical parts of the same leaf, and injects six biological replicates by exchanging the positions, and marks the injection areas.

[0055] (6) After the tobacco leaves were cultured at normal room temperature for 48 hours after injection, the injected leaves were immersed in 150 μg / mL luciferase substrate solution (D-Luciferin, Potassium Salt D-luciferin potassium salt) for 15 min, and LUC fluorescence was observed using a multifunctional imaging system.

[0056] Simultaneously, the detection was performed using a dual-luciferase reporter gene assay kit (Yisheng Biotechnology Co., Ltd., Shanghai). The specific steps are as follows:

[0057] (1) Take the leaf from the injection area and place it in a 2mL EP tube, add two small steel balls, freeze it quickly in liquid nitrogen, and grind the sample into powder using a tissue homogenizer (30Hz, 60s).

[0058] (2) After the crushing is completed, add 300 μL of lysis buffer to the EP tube, shake to mix, and let stand on ice for 5 min to allow the blade to fully decompose.

[0059] (3) Centrifuge at 12000 rpm for 3 min, and transfer the supernatant to a new EP tube for later use.

[0060] (4) Add 50 μL of supernatant to the culture plate, and set up 3 wells for each sample for replication.

[0061] (5) Prepare firefly luciferase (LUC) reaction solution and kidney luciferase (REN) reaction solution separately, i.e., dilute firefly luciferase substrate (50×) and kidney luciferase substrate (50×) to 1× working solution with the corresponding buffer solution. Incubate at room temperature.

[0062] (6) Add 100 μL of firefly luciferase reaction solution to the sample, shake to mix, and use a microplate reader (Varioskan LUX) to detect the activity of firefly luciferase (electrochemiluminescence, 1000 ms).

[0063] (7) Add 100 μL of Renali luciferase reaction solution, mix gently, and detect the activity of Renali luciferase. The enzyme activity detection should be completed within 30 minutes if possible.

[0064] (8) Analyze the data.

[0065] 4. Development of dCAPS molecular markers

[0066] For the two SNPs in the promoter region of the ZmEnd1 gene used to classify haplotypes, suitable restriction endonucleases were selected by analyzing their sequences using the dCAPS Finder 2.0 website (http: / / helix.wustl.edu / dcaps / dcaps.htmL). Specific primers were designed based on the selected endonuclease sequences. Mutations were introduced at appropriate positions in the primer design to ensure that the amplified product could be cleaved by the specific restriction endonuclease, while the other haplotype could not be cleaved. Using DNA from inbred lines of different haplotypes as templates, PCR reactions were performed using Phanta Max Super-Fidelity DNA Polymerase, and the results were detected by 1% agarose gel electrophoresis. The specific primers dCAPS-F / R designed in this invention are detailed in Table 1. The PCR products were digested with the selected restriction endonuclease (digestion system: 1 μL 10× QuickCut Buffer, 1 μL DNA, 1 μL QuickCut enzyme, 7 μL sterile water) at 37°C for 15 min. The banding patterns were detected and observed using agarose gel electrophoresis. Different haplotypes produced different band combinations due to differences in their SNPs, thus enabling genotype differentiation. Fragment amplification and enzyme digestion were performed on inbred lines of multiple different haplotypes, and the results of dCAPS molecular markers were compared with known genotype data (such as Sanger sequencing results) to confirm the effectiveness and accuracy of dCAPS molecular markers.

[0067] dCAPS molecular marker (SEQ ID NO.2, negative sense chain):

[0068] (The underlined part indicates the SNP-4-245033215 position (negative sense strand), the wavy line indicates the primer design site, and the lowercase letters cagtgg indicate the restriction enzyme site. If the restriction enzyme produces two bands, it is haplotype Hap1; if it cannot be cleaved and only produces one band, it is haplotype Hap2.)

[0069] The nucleotide sequence of the ZmEnd1 gene promoter (SEQ ID NO.3):

[0070] R represents A or G; a single underscore corresponds to SNP-4-245033215, and a double underscore corresponds to SNP-4-245033385.

[0071] 5. Spatiotemporal expression pattern analysis and subcellular localization of ZmEnd1

[0072] Analysis of expression patterns in different tissues: To further investigate the expression of ZmEnd1 in various tissues of maize, this invention collected three biological replicates each of root, stem, leaf, female ear, male ear, and 8-DAP to 21-DAP grain samples from the grain-filling stage of maize inbred line B73. RNA was extracted and reverse transcribed for qRT-PCR analysis. Primers are detailed in Table 1. The ZmActin1 gene was used as an internal control, and 2... -ΔΔCT The method calculates the relative expression level of the target gene.

[0073] Subcellular localization: To verify the subcellular localization of the ZmEnd1 gene, the ZmEnd1 gene was cloned into the pCAMBIA2300-eGFP vector to generate the p35S:ZmEnd1-eGFP fusion expression vector. Primers are detailed in Table 1. The fusion expression vector was then transformed into Agrobacterium strain GV3101, and the culture was cultured to OD200. 600 Agrobacterium suspension was prepared at a concentration of 0.8 and injected into tobacco leaves for transient expression. The p35S:eGFP vector was used as a negative control. After 48 h of culture, the eGFP fluorescence signal in tobacco leaves was detected by confocal fluorescence microscopy (Zeiss LSM 800, Baden-Württemberg, Germany).

[0074] 6. Creation of genetic material for ZmEnd1

[0075] The two maize ZmEnd1 gene EMS premature termination mutants of this invention were obtained from the maize EMS mutant website (http: / / www.elabcaas.cnnmemd / public / index.htmL), with the B73 inbred line as the wild type. DNA was extracted from the mutant lines using a modified SLS method. Specific detection primers were designed based on the mutation site location. Fragments containing the mutation site were amplified using the mutant DNA as a template, and sequencing was performed to determine if they were positively homozygous. The detection primers are detailed in Table 1.

[0076] 7. Determination of starch content in ZmEnd1 mutant grains

[0077] The starch content of seeds from ZmEnd1 mutant and wild-type individuals was determined using the Megazyme K-TSTA Total Starch Assay Kit. Five seeds were randomly selected and weighed. Sample pretreatment: The seeds were milled at 60Hz for 60 seconds and passed through a 100-mesh sieve. Five 100mg samples (accurately weighed) of the milled sample were taken from each sample and placed in 2mL centrifuge tubes. 0.2mL of 80% (v / v) ethanol aqueous solution was added, and the mixture was thoroughly mixed on a vortex mixer to disperse the sample. Immediately afterwards, 2mL of dimethyl sulfoxide (DMSO) was added to the centrifuge tubes, and the mixture was vortexed again. Preliminary incubation and enzymatic digestion: The centrifuge tubes were placed in a boiling water bath for 5 minutes. After removal, 3mL of heat-stable α-amylase (using 50mM MOPS buffer containing 5mM calcium chloride, pH 7.0, diluted 30 times) was immediately added to the centrifuge tubes, and the mixture was vortexed for 20 seconds. Return the centrifuge tubes to the boiling water bath and continue incubation for 6 minutes, vortexing at 2, 4, and 6 minutes to ensure homogeneity. Further enzymatic digestion and transfer: Transfer the centrifuge tubes to a 50°C water bath, add 4 mL of 200 mM sodium acetate buffer (pH 4.5) containing 5 mM calcium chloride, and then add 0.1 mL of amylase (AMG, 3,300 U / mL). Vortex the contents of the centrifuge tubes and incubate at 50°C for 30 minutes. Volumetric centrifugation and absorbance measurement: Transfer all reaction solutions to 100 mL volumetric flasks (using a funnel), and rinse the centrifuge tubes to remove any residue. Volume up to the mark using 200 mM sodium acetate buffer (pH 4.5) containing 5 mM calcium chloride and mix well. Take 2.0 mL from each sample solution, transfer to a microcentrifuge tube, and centrifuge at 13,000 rpm for 5 minutes. Prepare two aliquots of each sample. Accurately pipette 0.1 mL of the supernatant into the bottom of a 16 × 120 mm glass test tube. Add 3.0 mL of GOPOD reagent to each glass test tube and incubate in a 50 °C water bath for 20 min. Finally, measure the absorbance at a wavelength of 510 nm, using the reagent blank as a reference.

[0078] Starch content calculation:

[0079]

[0080] Wherein, ΔA refers to the absorbance of the sample solution minus the absorbance of the background blank sample solution; F is the conversion factor for converting the absorbance value to glucose in μg (100 μg glucose divided by the absorbance value measured by 100 μg glucose); EV is the sample extraction volume of 100 mL; 0.1 represents the volume of the sample analyzed; D is the sample solution dilution factor; 1 / 1000 represents the conversion from μg to mg; 100 / W represents the conversion to 100 mg sample, where W is the sample weight (mg); 162 / 180 is the final conversion factor for the conversion from free glucose to glucose present in starch.

[0081] 8. Determination of glucose-1-phosphate (G-1-P) content in ZmEnd1 mutant grains

[0082] The determination of glucose-1-phosphate content in the seeds was performed using a kit from Shanghai Tongwei Biotechnology Co., Ltd. The specific steps are as follows: Five groups of seeds were weighed for each sample, with 0.1 g of seed powder in each group. 1 mL of extraction buffer was added, and the mixture was homogenized in an ice bath. The mixture was centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was collected, and the corresponding reagents were added sequentially to a 96-well plate (Table 2).

[0083] Table 2 Mixed system for glucose-1-phosphate determination

[0084]

[0085] Calculation results: 1PG / G1P content (μg / g) = 836 × ΔA ÷ W × D, where D is the dilution factor (1 for undiluted samples) and W is the sample mass.

[0086] 9. Determination of uridine diphosphate glucose (UDPG) and trehalose-6-phosphate synthase (T-6-P) content in ZmEnd1 mutant grains

[0087] The content of uridine diphosphate glucose (UDPG) and trehalose-6-phosphate synthase (T-6-P) in corn kernels was determined using an ELISA kit from Shanghai Tongwei Biotechnology Co., Ltd. The procedure was as follows: Accurately weigh 0.1 g of corn kernel powder, add 1 mL of pre-cooled extraction buffer, homogenize in an ice bath, and then centrifuge at 12,000 × g at 4 °C for 10 min. Collect the supernatant. Remove the microplate from the aluminum foil bag after equilibration at room temperature. Add 50 μL of different concentrations of trehalose-6-phosphate and UDPG standard solutions sequentially to the standard wells. Add 10 μL of the sample to the sample wells and then add 40 μL of sample diluent. Do not add any liquid to the blank wells. Add 100 μL of trehalose-6-phosphate and UDPG-specific HRP-labeled antibodies to all wells except the blank wells. Seal the plate and incubate at 37 °C for 60 min. Discard the liquid in the wells, pat dry with absorbent paper, add washing buffer, and let stand for 1 min. Repeat the washing process 5 times. Add 50 μL of substrate A and 50 μL of substrate B to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution. Immediately measure the OD value using a microplate reader at a wavelength of 450 nm. Plot a linear regression curve between the concentration of the sea standard and the OD value using Excel, and calculate the content of trehalose-6-phosphate and uridine diphosphate glucose in the sample based on the equation.

[0088] 10. Scanning electron microscopy observation of the ZmEnd1 mutant

[0089] Grains from ZmEnd1 mutants (15DAP, 20DAP, and 25DAP) and wild-type samples were collected. Intact grains were removed from the ears using a blade and fixed in electron microscopy fixative for 24 hours. The fixed grain samples were then rinsed three times (15 min each) with 0.1 M phosphate buffer (pH 7.0). Subsequently, they were fixed in 1% osmium tetroxide solution (phosphate buffer) at room temperature for 2 hours, followed by three more rinses with phosphate buffer (15 min each). The samples were then sequentially dehydrated in solutions containing 30%, 50%, 70%, 80%, and 90% ethanol for 20 min each, and finally dehydrated twice (20 min each) with anhydrous ethanol. The samples were then permeated in a mixture of ethanol and isoamyl acetate in equal proportions for 30 min, followed by treatment with pure isoamyl acetate for 20 min. After drying using a critical point dryer, the samples were fixed to the sample stage with conductive adhesive and sputtered with gold. Finally, the starch granules were observed and imaged using a scanning electron microscope (SEM).

[0090] 11. In situ hybridization observation of ZmEnd1 mutant seeds

[0091] Sample fixation and dehydration: ZmEnd1 mutant and wild-type seeds 15 days after pollination were selected and fixed overnight at 4°C using pre-cooled 70% FAA solution. The samples were washed three times with 70% ethanol for 30 min each time to remove the fixative. Dehydration was performed on ice using a gradient of 30%, 50% (30 min each), 70%, 85%, and 100% ethanol (1 h each). After dehydration, the samples were stored overnight in anhydrous ethanol at 4°C. Then, they were infiltrated for 60 min each with 50-100 mL of xylene-ethanol solutions of different dilutions (25%, 50%, 75%, and 100%). Finally, the infiltration was repeated twice in 100% xylene. Paraffin embedding and sectioning: At 60°C, infiltrate the tissue with paraffin / xylene solutions of different dilutions (25%, 50%, 75%, 100%), using 50-100 mL for each concentration, for 2 hours each. Incubate the tissue overnight at 60°C with 100% paraffin. To prevent moisture penetration, ensure the beaker containing the tissue is well-sealed. Transfer the tissue to a mold, pour in molten paraffin, adjust the tissue position, and add more molten paraffin. After cooling, place the mold on ice for 5-10 seconds to allow the stainless steel embedding cassette to shrink. Then, remove the paraffin block with a scraper and cut sections to a thickness of 8-10 μm using a microtome.

[0092] RNA hybridization and imaging: Place the sections in working solution containing 10 μg / mL proteinase K and digest for 10-15 min at room temperature. This step helps improve probe penetration. Wash the sections twice with PBS for 5 min each time, then fix with 4% paraformaldehyde solution for 10 min at room temperature to inactivate proteinase K. Cover the sections with prehybridization buffer (containing 50% formamide, 10% dextran sulfate, 1×SSC, 0.1% Tween-20, 50 μg / mL heparin, and 50 μg / mL yeast tRNA) and incubate at 42°C for 1 h to reduce nonspecific binding. Add the labeled RNA probe (usually digoxigenin or biotin-labeled) to the hybridization buffer to a final concentration of approximately 1 ng / μL. Drop the hybridization solution onto the sections, cover with a coverslip, place in a humidified chamber, apply the probe to the tissue sections, and incubate overnight at 50°C. After hybridization, the sections were washed with 0.2×SSC and treated with RNase. They were then incubated with anti-digoxigenin antibody at room temperature for 2 hours, followed by washing with buffer containing 1% bovine serum albumin. The sections were then incubated with freshly dissolved NBT / BCIP solution for 12-15 hours and observed under a microscope.

[0093] 12. Rice heterologous expression of the ZmEnd1 gene

[0094] To further investigate the gene function of ZmEnd1, particularly its impact on crop yield composition and grain starch synthesis, ZmEnd1 was heterologously expressed in the superior rice inbred line Nipponbare (Nip). Using the pCUB vector as the backbone, Bar as the selection marker, and BamH1 (GGATCC) as the restriction enzyme site, the complete CDS sequence of ZmEnd1 was cloned into the vector via homologous recombination to form the pCUB:ZmEnd1 recombinant plasmid. The recombinant plasmid was transformed into Nip using Agrobacterium-mediated transformation, and ZmEnd1 overexpression-positive lines were obtained by PCR detection. After propagation to the T2 generation and positive PCR detection, the expression level of ZmEnd1 in the grains of each transgenic line was semi-quantitatively detected, and phenotypic identification was performed. Primers used for vector construction are detailed in Table 1.

[0095] II. Experimental Results

[0096] 1. Identification of the key gene ZmEnd1 controlling starch content in maize kernels using GWAS.

[0097] One significant SNP marker controlling corn starch content (SNP-4-245041154, P = 5.45E) was identified using GWAS in four environments (Xishuangbanna Dai Autonomous Prefecture, Yunnan Province (2020XSBN), Chongzhou City, Sichuan Province (2021CZ), Ya'an City, Sichuan Province (2021YA), and Xishuangbanna Dai Autonomous Prefecture, Yunnan Province (2021XSBN)) and under BLUP conditions. -16 ()( Figure 1 (a) and (b)). Using a 50 kb range upstream and downstream as the candidate gene screening interval, a total of 8 candidate genes were identified. Expression pattern analysis of these 8 genes revealed that Zm00001d054004 was highly expressed in the endosperm. Figure 1 In (c), it was named ZmEnd1 and further verified.

[0098] 2. Identify the genetic variation sites of ZmEnd1 affecting grain starch content using candidate gene association analysis.

[0099] This invention combines the starch content of grains from four environments and BLUP with candidate gene association analysis of the Zm00001d054004 gene promoter and gene ontology variant sites. Three significant SNPs were detected in the 2021YA, 2021CZ, and BLUP environments. Figure 2(a) A SNP (SNP-4-245029222) was identified in exon 5, resulting in a synonymous mutation (TCG / TCC) in serine (Ser); SNP-4-245033385 and SNP-4-245033215 are both located in the promoter region. SNP-4-245033385 was significantly associated with starch content in the 2021YA, 2021CZ, and BLUP environments, while SNP-4-245033215 was significantly associated with the starch content phenotype in 2021CZ. Figure 2 (a) Based on two SNPs, SNP-4-245033385 and SNP-4-245033215, 285 inbred lines were divided into two main haplotypes, Hap 1 (AA) and Hap2 (GG). In the 2021CZ, 2021YA, and BLUP environments, the grain starch content of Hap1 was significantly higher (P<0.001) than that of Hap2 (AA). Figure 2 (b)-(d)), therefore Hap1 was identified as the superior haplotype for maize kernel starch content. Quantitative determination of Hap1's LUC / REN value using a dual-LUCiferase Reporter Assay further confirmed that Hap1's promoter activity was significantly higher than Hap2's. Figure 2 (e) and (f)).

[0100] Specific primers were designed for the SNP-4-245033215 marker, introducing the restriction enzyme site Nco1. Genomic DNA from different haplotype materials was used as templates to amplify fragments (Hap1: B73, SCL16, SCL33, SCL116, SCL138; Hap2: SCL6, SCL90, SCL114, SCL128, SCL133). The amplified products were then digested with the Nco1 restriction endonuclease. Due to the introduction of the restriction site, a recognition site appeared in Hap1, leading to cleavage, while Hap2 remained undigested. In gel electrophoresis, the cleaved Hap1 gene fragment showed two bands. Results from multiple haplotype materials demonstrate that this dCAPS molecular marker can identify different haplotypes. Figure 3 ).

[0101] 3. Expression Pattern Analysis of ZmEnd1

[0102] To investigate the changes and differences in ZmEnd1 expression levels at different parts and developmental stages of grains, quantitative analysis was performed. The results showed that ZmEnd1 was highly expressed in grains at 10, 12, 14, and 21 days post-pollination, while expression levels were lower in root, stem, and leaf tissues. In grains, expression gradually increased with developmental time, peaking at 12 days post-pollination and decreasing during the grain-filling stage until grain maturity. Figure 4 (c) It was also found that ZmEnd1 was highly expressed in the endosperm of the grains, with a trend of high expression in the early stage of endosperm development, low expression at 10-20 DAP after pollination, followed by high expression again, reaching its highest level at 34 DAP, and relatively stable expression in the embryo. Figure 4 (a)). Furthermore, this invention found that the expression level of ZmEnd1 in Hap1 was significantly higher than that in Hap2 at different developmental stages of the grain. Figure 4 (d) Additionally, in situ hybridization results showed that ZmEnd1 was highly expressed mainly in the embryo and endosperm of 15 DAP seeds after pollination, consistent with the expression level. Figure 4 (b)

[0103] 4. Subcellular localization analysis of ZmEnd1 encoded protein

[0104] like Figure 5 As shown in (a), to clarify the expression of ZmEnd1 in subcellular structures, this invention constructed a pCAMBIA1305-35S-eGFP-ZmEnd1 fusion expression vector, using a non-fusion eGFP expression vector as a negative control. Figure 5 As shown in (b), after transformation of tobacco leaves, observation under a laser confocal microscope revealed that the ZmEnd1 protein was specifically expressed in the cell nucleus and cell membrane. Figure 5 As shown in (c), autofluorescence detection of chloroplasts revealed that the ZmEnd1 protein was not expressed in chloroplasts. These results indicate that the ZmEnd1 protein is located in the nucleus and cell membrane.

[0105] 5. Mutations in ZmEnd1 reduce starch synthesis in grains.

[0106] To investigate the impact of loss of function of the ZmEnd1 gene on maize kernels, two STOP-GAINED mutants of the ZmEnd1 gene, zmend1-1 and zmend1-2, were constructed using the B73 inbred line as a background. Ear length, ear diameter, 100-kernel weight, kernel length, and kernel width were measured in both EMS mutant lines zmend1-1 and zmend1-2, as well as in the wild-type B73 line. Statistical results showed that the 100-kernel weight and kernel length of mutants zmend1-1 and zmend1-2 were significantly lower than those of the B73 inbred line, while ear length and kernel thickness were also lower. There were no significant differences in kernel width and ear diameter between mutants zmend1-1 and zmend1-2 and the B73 inbred line. Figure 6 ).

[0107] In plant library tissues, UDP-Glc pyrophosphorylase (UGPase) converts UDP-Glc produced by SuSy into glucose-1-P to meet metabolic needs. The contents of uridine diphosphate glucose (UDPG), glucose-1-phosphate (G-1-P), starch, and trehalose-6-phosphate (T-6-P) in seeds of B73 and zmend1-1, zmend1-2 mutants were measured. The results showed that compared to wild-type B73, the mutants had significantly lower starch content and G-1-P, while UDPG was significantly increased. Figure 7 (a)-(c)); Trehalose-6-phosphate (T-6-P) showed no significant difference compared to the wild type. Figure 7 (d)

[0108] The above results indicate that the ZmEnd1 gene is involved in the biological pathway regulating starch synthesis in maize kernels. Loss of this gene function weakens the UDPase-catalyzed conversion of UDPG and pyrophosphate to G-1-P and uridine triphosphate (UTP), thus hindering the synthesis of downstream products G-1-P and the final product starch. Simultaneously, the upstream product UDPG accumulates due to reduced conversion caused by the weakened reaction, resulting in a significantly higher UDPG content in the mutant compared to B73. Furthermore, this indicates that mutations in the ZmEnd1 gene do not affect the accumulation and metabolism of intermediate products in other synthetic pathways.

[0109] 6. Scanning electron microscopy observation of ZmEnd1 mutant seeds

[0110] Scanning electron microscopy was performed on the grains of mutants zmend1-1 and zmend1-2 and wild-type B73 at 15 DAP, 20 DAP, and 25 DAP after pollination. The results showed that as grain filling progressed, the number and size of starch granules in B73 grains were significantly higher than those in the mutants. Figure 8At 25 days post-exposure (DAP), B73 showed relatively mature, polygonal, and regular starch granules, which were fully filled with a protein protective structure formed by gliadin. In contrast, the starch granules in the mutant were still developing ellipsoidal or irregular polygonal shapes, with many gaps within the cells. Therefore, this indicates that the mutation of the ZmEnd1 gene leads to smaller starch granule volume and delayed development in maize kernels.

[0111] 7. Heterologous expression of the ZmEnd1 gene in rice promotes the biosynthesis of starch content in grains.

[0112] To further investigate the gene function of ZmEnd1, particularly its impact on crop yield composition and grain starch synthesis, ZmEnd1 was heterologously expressed in the superior rice inbred line Nipponbare. After propagation to the T2 generation and positive PCR detection, the expression level of ZmEnd1 in the grains of each transgenic line was semi-quantitatively determined. Figure 9 As shown in (b), the expression level of the ZmEnd1 gene in the transgenic lines was significantly higher than that in the wild-type Nip.

[0113] like Figure 9 As shown in (c), the thousand-grain weight of the transgenic lines was significantly higher than that of the wild-type Nip; Figure 9 As shown in (a), (d) and (e), the starch content and G-1-P content of rice grains were significantly higher than those of wild-type Nip.

[0114] Experimental results show that heterologous expression of the ZmEnd1 gene in rice can promote the synthesis of G-1-P by increasing the expression level of UDP-glucose pyrophosphorylase (UDPase) in the starch synthesis pathway, thereby increasing starch accumulation and ultimately resulting in an increase in the thousand-grain weight of rice.

[0115] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a molecular marker in identifying the starch content trait of maize kernels, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

3. The molecular marker includes two SNP sites, SNP-4-245033215 and SNP-4-245033385. SNP-4-245033215 is located at the 1751st base of the nucleotide sequence, and SNP-4-245033385 is located at the 1921st base of the nucleotide sequence. The starch content of corn kernels with haplotype AA is higher than that of haplotype GG.

2. A method for identifying the starch content trait in corn kernels, characterized in that, Includes the following steps: Genomic DNA was extracted from the sample to be tested; Using the genomic DNA as a template, the molecular marker described in claim 1 is amplified to obtain the haplotype of the sample to be tested, and the starch content trait of corn kernels is determined: the starch content of corn kernels with haplotype AA is higher than that of haplotype GG.

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