A corn lodging-resistant molecular marker and application thereof

Abstract

The application discloses a corn lodging-resistant molecular marker and application thereof. The molecular marker SNP-1642 is a base substitution of "C / T" at the 1642th bp in the Zma-miR528b gene promoter region, the miR528 has a higher expression amount in the inbred line with the SNP-1642 genotype of T, and is accompanied by a higher lodging index, and the miR528 has a lower expression amount in the inbred line with the genotype of C, and is accompanied by a lower lodging index. Based on the single nucleotide polymorphism at the site, a restriction enzyme digestion amplification polymorphic sequence is developed. The molecular marker provided by the application can screen new corn varieties resistant to lodging through different haplotype nucleotide sequences, and has important application value for improving the lodging resistance of corn.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a molecular marker for lodging resistance in maize based on functional SNPs in the promoter region of the Zma-miR528b gene and its application. Background Technology

[0002] corn( Zea mays L As my country's largest grain crop, lodging has become a bottleneck restricting high and stable yields and mechanized harvesting. Lodging not only leads to a 10-30% yield loss but also significantly increases harvesting costs and the risk of grain mold. Traditional breeding relies on visual inspection in the field and manual selection, which is inefficient and time-consuming, making it difficult to meet the urgent needs of modern dense-planting, high-yield cultivation to resist lodging.

[0003] Previous studies have shown that plant microRNAs participate extensively in cell wall synthesis, lignin deposition, and mechanical strength formation by regulating target genes post-transcriptionally. miRNA528 is a small RNA specific to certain monocotyledonous plants. In plants such as *Brachys bipinnatifida*, *Alfalfa*, rice, and sorghum, the miR528 family consists of a single member; in maize, the miR528 family contains two members, miR528a and miR528b, but their mature sequences are identical. Studies have found that ZmmiR528 in maize can target the key genes ZmLAC3 and ZmLAC5 in the lignin synthesis pathway; in ZmmiR528 overexpression plants, the expression levels of ZmLAC3 and ZmLAC5 decreased, along with reduced lignin content and stem puncture strength; ZmmiR528 knockdown and ZmLAC3 overexpression plants exhibited opposite phenotypes, namely, increased lignin content and stem puncture strength. However, systematic mining of functional SNPs in the Zma-miR528 gene region in maize is currently lacking, and linkage or functional markers that can be directly used for molecular breeding are also scarce. Therefore, developing precise molecular markers based on functional SNPs of the Zma-miR528 gene is of great significance for shortening the breeding cycle of maize lodging resistance and improving selection efficiency. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular marker for lodging resistance in maize based on a functional SNP in the promoter region of the Zma-miR528b gene and its application.

[0005] A molecular marker for lodging resistance in maize, wherein the molecular marker is SNP-1642, is a "C / T" base substitution occurring at the 1642th bp of the promoter region of the Zma-miR528b gene; maize with the SNP-1642 genotype T has a higher lodging index, while maize with the SNP-1642 genotype C has a lower lodging index.

[0006] The nucleotide sequence of the promoter region of the Zma-miR528b gene is shown in SEQ ID NO: 1.

[0007] The nucleotide sequences of the sequencing primers are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0008] The nucleotide sequences of the enzyme digestion primers are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0009] A gene chip comprising sequencing primers shown in SEQ ID NO: 4-5.

[0010] The application of the aforementioned molecular markers for lodging resistance in the breeding of lodging-resistant maize varieties.

[0011] The beneficial effects of this invention are as follows: The molecular marker SNP-1642 of this invention involves a "C / T" base substitution at 1642 bp in the promoter region of the Zma-miR528b gene. In inbred lines with the SNP-1642 genotype T, miR528 expression is high, accompanied by a high lodging index, while inbred lines with the C genotype show low miR528 expression, accompanied by a low lodging index. Based on the single nucleotide polymorphism (SNP) at this site, a CAPS (capillary enzyme-amplified polymorphic sequence) was developed. The molecular marker provided by this invention can screen for lodging-resistant maize varieties using different haplotype nucleotide sequences, which has significant application value in improving maize lodging resistance. Attached Figure Description

[0012] Figure 1 The frequency distribution of the lodging index of 443 maize inbred lines is shown in the figure.

[0013] Figure 2 Association analysis of nucleotide sequence polymorphisms and lodging index in the promoter and gene regions of Zm-miR528a(A) and Zm-miR528b(B).

[0014] Figure 3 The relationship between the genotype of the SNP-1642 site in the Zm-miR528b promoter region and the lodging index and miR528 expression level; (A) Box plot of lodging index of materials with SNP-1642 genotype T and C in 264 maize materials; (B) Relative expression level of mature miR528 sequence in the stems of materials with SNP-1642 genotype T and C in 71 maize materials; (C) Box plot of lodging index of materials with SNP-1642 genotype T and C in 71 maize materials.

[0015] Figure 4The Zm-miR528b promoter region contains the SNP-1642 site sequence. TspR I restriction endonuclease agarose gel electrophoresis image. Detailed Implementation

[0016] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0017] Example 1: Investigation of lodging index of maize inbred lines

[0018] Forty-three maize inbred lines were planted on May 13, 2020, at the Shunyi Agricultural Experiment Station of the Institute of Crop Science, Chinese Academy of Agricultural Sciences (40.13°N, 116.65°E). The soil type of the experimental field was typical alkaline (pH 8.2) brown soil. Soil composition analysis showed that per kilogram of dry soil, there were 18.8 g of organic matter, 85.79 mg of available nitrogen (N), 40.7 mg of available phosphorus (P), and 151.2 mg of available potassium (K). Before sowing, 750 kg of compound fertilizer (15 N-15 P2O5-15 K2O) was applied per hectare as basal fertilizer. At the tasseling stage of maize, 450 kg of urea (N≥46.4%) was applied per hectare as top dressing. The experiment used an α(0,1) Latin square design, randomly arranged, with each material sown in two rows, 3 m long, 60 cm row spacing, and 25 cm plant spacing, with two replicates. Irrigation was carried out on May 20th and June 20th, 2020, with each irrigation being 70mm. The experimental field was hit by a storm in July 2020, causing varying degrees of lodging in the maize inbred lines. The field lodging resistance of 443 maize inbred lines under natural conditions was evaluated. Based on the proportion of lodged plants in each row, the maize lodging index was divided into four levels: Level 1 (lodging rate ≤10%), Level 2 (lodging rate 10%-50%), Level 3 (lodging rate 50-90%), and Level 4 (lodging rate ≥90%). The lodging index results for these materials are as follows: Figure 1 As shown.

[0019] Example 2: Analysis of Zm-miR528 gene sequence polymorphism in natural maize populations

[0020] Genomic DNA was extracted from 300 randomly selected materials from 443 maize inbred lines. The promoter and gene region sequences of Zm-miR528a and Zm-miR528b in maize inbred lines were amplified by PCR using primer pairs 5'-CTTCATTTCAAATGGTGTCCC-3' (SEQ ID NO: 2) and 5'-GTGGAATGGAATGCCAGCGATG-3' (SEQ ID NO: 3), and primer pairs 5'-GCTCTATAATTCTCTACTGCATCTG-3' (SEQ ID NO: 4) and 5'-GGTTGCGCTTCTGAGTTTTT-3' (SEQ ID NO: 5). Agarose gel electrophoresis revealed single bands for the Zm-miR528a and Zm-miR528b genes in 264 samples. First-generation sequencing was then performed on these bands to analyze sequence polymorphisms in the Zm-miR528a and Zm-miR528b genes in maize inbred lines, identifying 41 and 48 single nucleotide polymorphism (SNP) sites, respectively. Candidate gene association analysis for Zm-miR528a and Zm-miR528b was also conducted using the field lodging index of maize inbred lines. Results are shown below. Figure 2 Nucleotide sequence polymorphisms in the Zm-miR528a promoter region and gene region were not significantly associated with the maize stalk lodging index. Figure 2 A); The nucleotide sequence polymorphism at SNP-1642 (B73 RefGen_v4:Chr9:156645373) in the Zm-miR528b promoter region was significantly associated with the lodging index of maize stalks. Figure 2 B), suggesting that the SNP-1642 site located in the Zm-miR528b promoter region may be a key polymorphic site for stem lodging phenotypic variation.

[0021] Example 3: The correlation between the SNP-1642 site polymorphism in the Zm-miR528b promoter region and maize lodging.

[0022] In a population of 264 maize inbred lines, the inbred lines were divided into two haplotypes based on the different genotypes at the SNP-1642 site in the Zm-miR528b promoter region, and the differences in lodging index were compared. Among them, 253 inbred lines had the SNP-1642 genotype C, and 11 inbred lines had the SNP-1642 genotype T. Compared to the haplotype with SNP-1642 genotype C, the lodging index of the inbred lines with SNP-1642 genotype T was significantly increased. Figure 3A). From 264 materials, 10 materials with SNP-1642 genotype T and 61 materials with SNP-1642 genotype C were randomly selected. The expression level of mature miR528 in the stems of these materials was detected by stem loop Q-PCR. The results showed that the relative expression level of mature miR528 sequence in the inbred line material with SNP-1642 genotype T was significantly higher than that in the material with SNP-1642 genotype C. Figure 3 B); and in inbred lines with SNP-1642 genotype T, miR528 expression was higher, accompanied by a higher lodging index, while inbred lines with genotype C showed lower miR528 expression, accompanied by a lower lodging index (B). Figure 3 C).

[0023] Example 4: Development of CAPS marker at SNP-1642 site in Zm-miR528b promoter region

[0024] To rapidly detect the genotype of the SNP-1642 site in the Zm-miR528b promoter region, a restriction enzyme amplification polymorphic sequence (CAPS) was developed based on the single nucleotide polymorphism at this site. Five inbred lines with SNP-1642 genotype T and eight inbred lines with SNP-1642 genotype C were randomly selected. PCR amplification was performed using primer pairs 5'-AACGGTAGAGGTTCATCCA-3' (SEQ ID NO: 6) and 5'-CAGTCAAGTGGCATAGGC-3' (SEQ ID NO: 7) to obtain DNA sequences containing the SNP-1642 site. Then, 5 μL of the PCR product was added, along with 1 μL of the corresponding 10× restriction buffer and 1 μL of TspRI restriction endonuclease. The volume was brought to a final volume of 10 μL with ddH2O, and the mixture was incubated at 37°C for 2 hours for restriction digestion. After the enzyme digestion reaction, 5 μL of the original PCR solution and the digestion solution were respectively subjected to agarose gel electrophoresis. It was found that the PCR product with SNP-1642 genotype C could be degraded by TspRI endonuclease, while the PCR product with SNP-1642 genotype T could not be degraded by TspRI endonuclease. Figure 4 This indicates that using the CAPS marker can effectively distinguish lodging-resistant corn materials.

[0025] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of a maize anti-lodging molecular marker in the breeding of a maize variety resistant to lodging, characterized in that, The molecular marker is SNP-359, which is a base substitution of "C / T" at the 359th bp of the Zma-miR528b gene promoter region; the lodging index of the corn with the genotype T of SNP-359 is higher, and the lodging index of the corn with the genotype C of SNP-359 is lower; the nucleotide sequence of the Zma-miR528b gene promoter region is shown as SEQ ID NO:

1.

2. The enzyme and enzyme digestion primer combination for detecting the maize anti-lodging molecular marker as set forth in claim 1, characterized in that, The enzyme is TspRI restriction enzyme, and the nucleotide sequences of the enzyme cutting primers are shown as SEQ ID NO:6 and SEQ ID NO:7, respectively.

Images