Primer of molecular marker closely linked to maize ear shape flat and application

Abstract

The application relates to primers for amplifying a molecular marker closely linked to maize ear type flatness and application, and belongs to the technical field of biology. The molecular marker is located on the 4th chromosome of maize and is a molecular marker JX1. The primer sequence for amplifying the molecular marker JX1 is as follows: JX1-L: 5'-TCACAACTCTCCTCTTCGTCGT-3'; JX1-R: 5'-TTGGAGTAAACACAAGGAGGGT-3'. The primers for amplifying the molecular marker closely linked to maize ear type flatness are applied to maize ear type molecular marker assisted breeding, maize ear type development germplasm resource screening and maize ear type genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to primers and their applications for amplifying molecular markers closely linked to the flattening of maize ears. Background Technology

[0002] Corn is the world's highest-yielding food crop. Grain yield is determined by the number of ears per acre and the weight of grains per ear, with ear diameter (which determines the number of rows per ear) being the key factor, accounting for more than 30% of the yield.

[0003] Maize ear development depends on the precise regulation of the inflorescence meristem (IM), and abnormalities in its morphology directly lead to ear defects (such as flattened ears or disordered ear rows). This invention addresses... efd1 The mutant is a typical example: its dysregulation of IM stem cell activity leads to structural flattening (at the 4mm stage), ultimately resulting in disordered ear row count and reduced grain yield at maturity. Due to the complexity of multi-gene regulation, ear type development involves... ZAG1, TSH4, fea3 The cascade regulatory network of more than ten genes exhibits significant epistatic effects; however, quantitative traits are limited: ear traits are easily affected by environmental disturbances (phenotypic variation coefficient ≥20%), and traditional QTL mapping has low accuracy; genetic background interference exists: background noise exists in conventional populations (such as F2), and minor QTLs are easily masked (Chen et al., 2024; Yang et al., 2024). Gene mapping presents certain challenges.

[0004] In view of the above-mentioned bottlenecks, this invention is proposed.

[0005] This invention provides an efficient solution based on BSA initial localization and JX1 molecular markers: the target is achieved through JX1 markers (Chr. 4 9.42Mb). efd1 Rapidly pinpoint the mutation range; overcome interference from complex genetic backgrounds, precisely narrow the localization range from the initially localized range to the range usable for breeding, and improve the efficiency of ear-type molecular breeding. Summary of the Invention

[0006] The purpose of this invention is to identify a natural mutant with a flattened ear morphology and an increased number of ear rows. efd1 The molecular markers closely linked to the major QTLs of ear type in maize and their applications, which can be used for marker-assisted breeding of maize ear type.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] Primers for amplifying a molecular marker closely linked to maize ear flattening, the molecular marker being closely linked to a major QTL for maize ear flattening, located on maize chromosome 4, and being molecular marker JX1.

[0009] Furthermore, the molecular marker JX1 is located at chr4: 8242210-8242387. The physical location of this invention is referenced to version V4.

[0010] Furthermore, the primer sequences for amplifying the molecular marker JX1 are as follows:

[0011] JX1-L: 5'-TCACAACTCTCCTCTTCGTCGT-3' (Sequence 1);

[0012] JX1-R: 5'-TTGGAGTAAACACAAGGAGGGT-3' (sequence 2).

[0013] Furthermore, primers for amplifying molecular markers closely linked to maize ear flattening were applied in marker-assisted breeding of maize ear type, screening of germplasm resources for maize ear type development, and genetic improvement of maize ear type.

[0014] Furthermore, the method for identifying maize ear type traits in maize ear type genetic improvement includes the following steps:

[0015] Extracting genomic DNA from maize leaves;

[0016] Using maize leaf genomic DNA as a template, PCR amplification was performed using primers JX1-L / JX1-R;

[0017] Agarose gel electrophoresis was used to identify the PCR amplification results: when the molecular marker JX1 was detected to be 178 bp, it indicated that the spikelet type of the sample was normal; when the molecular marker JX1 was detected to be 157 bp, it indicated that the spikelet type of the sample was flat.

[0018] The PCR amplification system consists of 10 μL of components, including: 2 μL DNA, 0.5 μL each of the left and right primers, 5 μL 2x TaqMaster Mix (Novozymes P112), and 2 μL ddH2O.

[0019] The Touch Down PCR amplification program was used: 95℃ for 5 min; 95℃ for 30 s, 65℃ for 30 s, decreasing by 1℃ per cycle, 72℃ for 45 s, for a total of 8 cycles; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 45 s, for a total of 28 cycles; 72℃ for 10 min.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The molecular markers in this invention are closely linked to major QTLs for ear type development in maize. They can be applied to marker-assisted breeding of ear type, screening of germplasm resources for ear type development, and genetic improvement of maize ear type development. Attached Figure Description

[0022] Figure 1 for efd1 Comparison of ear type phenotype with Lx9801 mature ears;

[0023] Figure 2 for efd1 Stereoscopic and scanning electron microscopic images of female ear development compared to Lx9801;

[0024] Figure 3 For Lx9801 and efd1 middle fea2, fea3 Differential expression analysis of genes at the 4mm stage of female ear differentiation;

[0025] Figure 4 BSA analysis of the normal and flattened spikelet phenotypes in the F2 population at the 4mm stage of female spike differentiation;

[0026] Figure 5 for efd1 The finely mapped regions, combined with genotype and phenotype, will efd1 It is located within a physical region of 1.13 Mb on chromosome 4, which contains 7 candidate genes;

[0027] Figure 6 Agarose gel electrophoresis was used to locate the key marker JX1; 1 was a mixed pool of parental B73, and 2 was a mixed pool of parental... efd1 Mixed pool, 3 is a mixed pool with normal phenotype, 4 is a mixed pool with flat spikelet phenotype; the linkage verification of some markers is shown below. Detailed Implementation

[0028] The technical solution and effects of the present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0029] Example 1: efd1 Identification and observation of mutant ear phenotype

[0030] Experimental material treatment: using maize inbred line Lx9801 and its natural mutants. efd1The material used was the maize inbred line Lx9801, which is available to the public from the State Key Laboratory of Wheat and Maize Crop Science, College of Agriculture, Henan Agricultural University. It was planted in Zhongmu, Yuanyang, and Hainan in 2022 and 2023, respectively, using conventional field management (4 m row length, 0.65 m row spacing, 20 hills per row, single seed sowing). Materials were selected at the 2 mm and 4 mm ear development stages, and mature ears and plants were also collected.

[0031] Phenotypic observation methods:

[0032] Mature plant type and ear phenotype: Lx9801 and [other varieties] were photographed using a Nikon camera. efd1 Mature plants and fruit clusters were observed to differentiate plant type and fruit cluster morphology. Results are shown in […]. efd1 .

[0033] Observation of female ear development stages: The morphology of female ears at 2 mm and 4 mm was observed under a stereomicroscope; the female ears at 4 mm were observed under a scanning electron microscope (samples were fixed with 2 wt% glutaraldehyde, subjected to gradient dehydration, critical point drying, and gold sputtering, and then observed under a FEIQuanta 45 environmental scanning electron microscope with a working voltage of 10 kV and a vacuum degree of 5×10⁻⁶). -3 Pa), results are shown in Figure 1 .

[0034] See results Figure 2 and Figure 1 Compared to Lx9801, Figure 2 There was no significant difference in plant type at maturity, but the ears showed significant flattening and an increase in the number of ear rows; at 2 mm, the head of the female ear began to deform, and at 4 mm, the apical meristem (IM) became significantly flattened and the ear rows increased.

[0035] Example 2: efd1 Comparative analysis of the mutant with known spikelet development mutants

[0036] 2.1 Experimental Materials and Sample Collection: Wild-type maize (WT, represented by the maize inbred line Lx9801) and ear-flattening mutants were selected. efd1 As experimental materials, WT and [other samples were collected] at the critical stage when the maize female ear developed to 4 mm (this stage is the period of inflorescence meristem differentiation and spikelet arrangement determination, which plays a decisive role in ear shape formation). efd1 The female ear tissue of the plant was quickly frozen in liquid nitrogen and stored at -80°C for later use in gene expression analysis and sequence detection.

[0037] 2.2 RNA Extraction and cDNA Synthesis: WT and cDNA were extracted using the TRIzol method. efd1Total RNA from female ear tissue: Take 50 mg of frozen tissue, grind with liquid nitrogen, add 1 mL of TRIzol reagent (Invitrogen), shake thoroughly to mix, and let stand at room temperature for 5 min; add 200 μL of chloroform, shake vigorously for 15 s, and let stand at room temperature for 3 min; centrifuge at 4 ℃ and 12,000×g for 15 min, and transfer the upper aqueous phase to a new centrifuge tube; add an equal volume of isopropanol, and let stand at room temperature for 10 min; centrifuge at 4 ℃ and 12,000×g for 10 min, discard the supernatant, wash the precipitate with 75 wt% ethanol, air dry, and dissolve in 30 μL of RNase-free water.

[0038] 2.3 Known genes for spikelet development ( efd1 , fea2 Expression level analysis of )

[0039] Detection was performed using real-time quantitative PCR (qRT-PCR). fea3 , fea2 In WT and fea3 The relative expression level in [the sample / molecule]. Reverse transcription is performed to generate first-strand cDNA, which is then used as a template. efd1 The primer pair consisted of 5'-AACCGATTGTCCGGTTCAGT-3' (sequence 3) and 5'-CCTCCAAAGACCTGCACCC-3' (sequence 4). fea2 Real-time quantitative PCR amplification was performed using a primer pair consisting of 5'-GCCCGCGGGGTGTCA-3' (sequence 5) and 5'-CGGGAAGCAGCTGTAGAAAGACA-3' (sequence 6) to identify... fea3 as well as fea2 Relative gene expression levels. The Ubi gene was selected as the internal control gene (the primer pair used to identify the internal control gene consisted of 5'-GCTGCCGATGTGCCTGCGTCG-3' (sequence 7) and 5'-CTGAAAGACAGAACATAATGAGCACAG-3' (sequence 8)). Real-time quantitative PCR was performed using an Applied Biosystems 7500 RealTime PCR system (ABI, USA), with three replicates per experiment. Relative expression levels were calculated using the 2-ΔΔCT method.

[0040] See results fea3 By comparing wild-type WT and Figure 3 In the plant efd1 and fea2 The cDNA sequences and expression levels were compared, and no mutation sites were found, nor were the expression levels significantly different. This indicates that... fea3 It is a newly discovered spike flattening mutant.

[0041] Example 3: efd1 BSA-Seq initial localization analysis of mutants

[0042] 3.1 Experimental Materials and Population Construction: [The text abruptly ends here, likely due to an incomplete sentence or efd1 F1 was obtained by crossing B73 as the female parent and B73 as the male parent, and F2 population was constructed by self-pollination.

[0043] 3.2 DNA Extraction and Molecular Marker Development: Genomic DNA was extracted from individual leaves of the F2 population using the SLS method: 1 cm of leaf sample was taken. 2 Fresh leaves were ground with liquid nitrogen and then added to 500 μL of SLS extraction buffer (containing 0.2 M EDTA, 1 M Tris-HCl, 1 M NaCl, 1 wt% SLS, pH=8.0). The mixture was then mixed with an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), centrifuged at 12,000×g for 10 min, and the supernatant was precipitated with isopropanol, washed with 75 wt% ethanol, dissolved in deionized water, and stored at -20 ℃ for later use.

[0044] 3.3 BSA-Seq Analysis Method for DNA Pool Construction: Leaves from 50 extremely flattened ears and 50 normal ears in the F2 population were selected to construct mutant and normal pools, respectively. Simultaneously, DNA was extracted from... efd1 Parental DNA.

[0045] 3.4 Sequencing and Data Processing: 125 bp and 150 bp paired-end sequencing was performed on the Illumina platform. After filtering out low-quality reads, the clean reads were aligned to the maize B73 v4 reference genome (ftp: / / ftp.ensemblgenomes.org / pub / plants / release-41 / fasta / zea_mays / dna / ), and Q30, GC content, and alignment rate were calculated.

[0046] See results efd1 .Will Figure 4 Preliminary localization to the 4.68-11.26 Mb region (6.58 Mb) on chromosome 4.

[0047] Example 4: efd1 BSA-Seq fine localization analysis of mutants

[0048] 4.1 Experimental Materials and Population Construction: B73 was used as the donor parent. efd1 As recurrent parents, F1 was obtained through hybridization and then backcrossed to construct the BC1F1 population.

[0049] 4.2 DNA extraction and molecular marker development: Genomic DNA of BC1F1 population seeds was rapidly extracted using the alkaline boiling method: a small amount of seed aleurone layer tissue was taken, 70 μL of 0.1M NaOH solution was added, and the mixture was treated at 99 ℃ for 12 min. After cooling, 70 μL of TE buffer (containing 0.5 M EDTA and 1 M Tris-HCl) at pH=2.0 was added for neutralization. After standing at room temperature for 10 h, the mixture was used for PCR analysis.

[0050] 4.3 Molecular marker development: Based on BSA-Seq results, molecular markers were developed in the 4.68-11.26Mb region of chromosome 4, primers were designed (product length 150-200 bp), and polymorphism was verified by PCR amplification.

[0051] 4.4 PCR amplification system: The PCR amplification system is 10 μL, and the components include: 2 μL DNA, 0.5 μL each of the left and right primers, 5 μL 2x Taq Master Mix (Novozymes P112) and 2 μL dd H2O.

[0052] PCR amplification program: Using the Touch down PCR amplification program: 95℃ for 5 min; 95℃ for 30 s, 65℃ for 30 s, decreasing by 1℃ per cycle, 72℃ for 45 s, for a total of 8 cycles; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 45 s, for a total of 28 cycles; 72℃ for 10 min.

[0053] The amplification products were analyzed for genotype by 4wt% agarose gel electrophoresis.

[0054] 4.5 Screening of Recombinant Individual Plants: Genotypic analysis of the BC1F1 population was performed using polymorphic markers to screen for recombinant individual plants, and these plants were then combined with phenotypic analysis to determine their recombinant composition. efd1 The positioning narrows down the range.

[0055] See results efd1 and Figure 5 . Figure 6 It is located in the 8.29-9.42Mb region (1.13Mb) of chromosome 4, contains 7 genes, and the key molecular marker is JX1.

[0056] The primer sequences for amplifying the molecular marker JX1 are as follows:

[0057] JX1-L: 5'-TCACAACTCTCCTCTTCGTCGT-3' (Sequence 1);

[0058] JX1-R: 5'-TTGGAGTAAACACAAGGAGGGT-3' (sequence 2).

[0059] Example 5

[0060] Example 4 shows the amplification and maize ear-type development mutant. efd1 efd1 Application of primers for tightly linked molecular markers in marker-assisted breeding of maize ear type, screening of germplasm resources for maize ear type development, and genetic improvement of maize ear type.

[0061] The method for identifying the maize ear thickness trait in the application of maize ear type genetic improvement includes the following steps:

[0062] Extracting genomic DNA from maize leaves;

[0063] Using maize leaf genomic DNA as a template, PCR amplification was performed using primers JX1-L / JX1-R;

[0064] Agarose gel electrophoresis was used to identify the PCR amplification results: when the molecular marker JX1 was detected to be 178 bp, it indicated that the spikelet type of the sample was normal; when the molecular marker JX1 was detected to be 157 bp, it indicated that the spikelet type of the sample was flat.

[0065] The PCR amplification system consists of 10 μL of components, including: 2 μL DNA, 0.5 μL each of the left and right primers, 5 μL 2x TaqMaster Mix (Novozymes P112), and 2 μL ddH2O.

[0066] The Touch Down PCR amplification program was used: 95℃ for 5 min; 95℃ for 30 s, 65℃ for 30 s, decreasing by 1℃ per cycle, 72℃ for 45 s, for a total of 8 cycles; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 45 s, for a total of 28 cycles; 72℃ for 10 min.

[0067] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of molecular marker JX1 or primers amplifying said molecular marker JX1 in marker-assisted breeding of maize ear type, screening of maize ear type development germplasm resources, and genetic improvement of maize ear type, characterized in that, The molecular marker JX1 is located on chromosome 4 of maize, and the marker position of molecular marker JX1 is chr4: 8242210-8242387. This physical position is based on version V4. The primer sequences for amplifying the molecular marker JX1 are as follows: JX1-L: 5'-TCACAACTCTCCTCTTCGTCGT-3', as shown in Sequence 1; JX1-R: 5'-TTGGAGTAAACACAAGGAGGGT-3', as shown in Sequence 2; If the molecular marker JX1 size is 178bp, it indicates that the corn ear type is normal; if the molecular marker JX1 size is 157bp, it indicates that the corn ear type is flat; if the molecular marker JX1 size shows both 178bp and 157bp bands, it is a heterozygous case, which indicates that the corn ear type is normal.

2. A method for identifying ear type traits in maize ear type genetic improvement, characterized in that, Includes the following steps: Genomic DNA was extracted from maize leaves; Using maize leaf genomic DNA as a template, PCR amplification was performed using primers JX1-L / JX1-R; Agarose gel electrophoresis was used to identify the PCR amplification results: when the molecular marker JX1 was detected to be 178 bp, it indicated that the ear shape of the sample was normal; when the molecular marker JX1 was detected to be 157 bp, it indicated that the ear shape of the sample was flat; when both 178 bp and 157 bp bands of molecular marker JX1 were detected, it was a heterozygous situation, indicating that the ear shape of the maize was normal. The primer JX1-L: 5'-TCACAACTCTCCTCTTCGTCGT-3', as shown in sequence 1; The primer JX1-R: 5'-TTGGAGTAAACACAAGGAGGGT-3', as shown in sequence 2.

3. The method according to claim 2, characterized in that, The PCR amplification system is 10 μL, and its components include: 2 μL DNA, 0.5 μL each of the left and right primers, 5 μL 2x Taq Master Mix and 2 μL ddH2O.

4. The method according to claim 2, characterized in that, The Touch-down PCR amplification program was used: 95℃ for 5 min; 95℃ for 30 s, 65℃ for 30 s, decreasing by 1℃ per cycle, 72℃ for 45 s, for a total of 8 cycles; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 45 s, for a total of 28 cycles; 72℃ for 10 min.

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