SNP (Single Nucleotide Polymorphism) molecular marker related to drought resistance of corn and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to drought resistance of corn and application of the SNP molecular marker, and belongs to the technical field of biology. The SNP molecular marker is located at the 43089691 position of a chromosome 2 of a corn reference genome Maize B73 AGPv4, and the polymorphism of the SNP molecular marker is T / C. Through verification, when the nucleotide sequence position 2-43089691 is C, the yield indexes such as the single spike weight and the grain weight per spike of corn are remarkably improved, and the SNP molecular marker can be used for screening corn materials with drought resistance. The SNP molecular marker disclosed by the invention can also be used for corn molecular assisted breeding and high-quality germplasm resource identification, so that the development of a corn drought-resistant material and the breeding process of a new variety can be accelerated. Therefore, the invention provides theoretical basis and technical support for corn stress resistance molecular marker-assisted breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a SNP molecular marker related to drought resistance in maize and its application. Background Technology

[0002] Maize (Zea mays L.) is one of the world's major crops used for food, cash crops, and feed. Drought is one of the main abiotic factors causing crop yield reduction. For maize, seasonal drought stress before and after sowing, tasseling, and silking is particularly detrimental to yield. SNPs (single nucleotide polymorphisms) are important molecular genetic markers with the advantages of wide distribution and convenient detection, and are also of great significance for maize molecular breeding.

[0003] General Regulatory Factor (GRF) proteins, also known as 14-3-3 proteins, are a class of highly conserved molecular chaperone proteins in eukaryotes, widely distributed in various plant organelles and possessing important biological functions. They can specifically recognize phosphorylated target proteins and participate in processes such as plant growth and development, cell elongation and division, seed germination, vegetative growth, and reproductive growth. They also play a crucial role in plant responses to abiotic stresses such as salinity, osmotic stress, drought, and low temperature. During plant growth and development, GRF proteins regulate target gene expression and physiological processes by interacting with proteins and transcription factors in various hormone signaling pathways. For example, in rice, OsGF14f interacts with OsbZIP23, enhancing the transcriptional regulatory function of OsbZIP23, thereby increasing rice's tolerance to osmotic stress by activating the expression of downstream stress-response genes. Overexpression of the 14-3-3 genes BdGF14d and SiGRF1 in plants can enhance their salt tolerance. SOS is a key protein in the salt-sensitive pathway. Under normal growth conditions, SOS2 is phosphorylated by SOS2-like protein kinase 5 (PKS5). Arabidopsis thaliana's 14-3-3λ and 14-3-3κ reduce SOS2 phosphorylation by inhibiting the activity of the key kinase PKS5, thereby relieving the inhibition of SOS2 by the 14-3-3 protein and thus inhibiting the SOS pathway. Simultaneously, the reduction in PKS5 activity alleviates the inhibition of H... + -Inhibition of ATPase and promotion of H + -ATPase interacts with 14-3-3ω, activating H+ + -ATPase activity provides the driving force for SOS1. Simultaneously, salt stress can also activate calcium-dependent protein kinases (CDPKs), thereby phosphorylating vacuolar two-pore K+. +Channel 1 (TPK1). Furthermore, the 14-3-3 protein GRF6 interacts with phosphorylated TPK1 and enhances its activity, thereby promoting potassium ion outflow from the vacuoles to the cytoplasm and improving plant salt tolerance. In addition, GRF proteins regulate physiological processes such as stomatal opening and closing, root growth, and photosynthesis in response to drought stress through various transcription factors and signaling pathways. Overexpression of Arabidopsis GF14λ in cotton improves drought resistance, increases photosynthesis, and reduces wilting. Expression of ZmGF14-6 in rice enhances drought resistance by inducing the expression of drought-related genes in the roots. GRF proteins act as nodes in plant signal transduction networks, integrating different signaling pathways and coordinating growth and environmental adaptation.

[0004] Currently, there are no reports on whether there are SNP sites in GRF genes in maize that are significantly associated with drought. Summary of the Invention

[0005] The purpose of this invention is to provide a SNP molecular marker related to drought resistance in maize and its application, so as to solve the problems existing in the prior art. This invention provides an SNP molecular marker related to drought resistance in maize, providing a theoretical basis and technical support for marker-assisted breeding of maize stress resistance.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a SNP molecular marker associated with drought resistance in maize. The SNP molecular marker is located at chromosome 43089691 of the maize reference genome Maize B73 AGP_v4, and the polymorphism is T / C.

[0008] The present invention also provides a primer pair for amplifying the SNP molecular marker, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2.

[0009] The present invention also provides the application of the primer pair described herein in the preparation of a kit for identifying drought resistance in maize.

[0010] The present invention also provides a kit for identifying drought resistance in maize, comprising the primer pair described above.

[0011] Optionally, the kit also includes 2×Rapidtaq Master PCR mix.

[0012] This invention also provides the use of the SNP molecular marker, the primer pair, or the kit described herein in any of the following:

[0013] (1) Identify the drought resistance of corn;

[0014] (2) Identify and screen drought-resistant maize varieties or lines;

[0015] (3) Molecular marker-assisted breeding for drought resistance in maize;

[0016] (4) Improve drought-resistant maize germplasm resources;

[0017] (5) Identify and screen high-yielding maize varieties or strains.

[0018] This invention also provides a method for identifying the drought resistance of maize, comprising the following steps:

[0019] Using the genomic DNA of the maize sample to be tested as a template, PCR amplification was performed using the primer pair to obtain the amplification product; when the amplification product contains the amplification fragment with a nucleotide sequence as shown in SEQ ID NO.3, it indicates that the maize sample to be tested has high drought resistance.

[0020] Optionally, the PCR amplification reaction system is: 10 μL of 2×Rapid Taq Master PCR mix, 0.5 μL each of upstream and downstream primers, 1 μL of DNA template, and 8 μL of double-distilled water.

[0021] Optionally, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 8 s, 56℃ annealing for 8 s, 72℃ extension for 8 s, 40 cycles; 72℃ for 5 min.

[0022] The present invention discloses the following technical effects:

[0023] The SNP molecular markers provided by this invention are significantly correlated with yield traits (ear weight and grain weight) of maize plants under drought conditions. When the nucleotide sequence position 2-43089691 (at position 43089691 on chromosome 2 of the maize reference genome Maize B73 AGP_v4) is C, yield indicators such as ear weight and grain weight are significantly improved, indicating that these SNP molecular markers can be used to screen for drought-resistant maize materials. The SNP molecular markers of this invention can also be used for molecular-assisted breeding and identification of high-quality germplasm resources in maize, thereby accelerating the creation of drought-resistant maize materials and the breeding process of new varieties. Therefore, this invention provides a theoretical basis and technical support for marker-assisted breeding of maize for stress resistance. Attached Figure Description

[0024] 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.

[0025] Figure 1 The results show the sequence alignment and PCR amplification results in different maize materials; where a is the relationship between the SNP site and ear height analyzed based on data from the MaizeGDB webpage; b is the electrophoresis diagram of PCR amplification using DNA from 18 different maize inbred lines as templates, where M represents Marker (DL2000); c is the sequence alignment analysis diagram of DNA from 18 different maize inbred lines.

[0026] Figure 2 The graph shows the differences in yield traits among 18 different maize inbred lines. Specifically, a is a schematic diagram of the ear phenotype of the 18 different maize inbred lines (bar = 5 cm); b is a statistical graph of single ear weight traits among two different SNP-type maize inbred lines; c is a statistical graph of ear grain weight traits among two different SNP-type maize inbred lines; WW: normal irrigation group; WS: drought and water shortage group. *** This represents P < 0.001; **** This means P < 0.0001. Detailed Implementation

[0027] 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.

[0028] 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 in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] The different maize inbred lines used in the embodiments of this invention were all collected and preserved by Sichuan Agricultural University. The 18 different maize inbred lines used in the following embodiments are shown in Table 1, all provided by the germplasm resource bank of the Maize Research Institute of Sichuan Agricultural University.

[0033] Table 1. Counting data from 18 different maize inbreeding systems

[0034]

[0035] Example 1

[0036] Sequence alignment analysis using the Maize GDB database revealed a SNP mutation in the CDS region of the maize ZmGRF24 gene, located at position 43089691 on chromosome 2 (a T / C variant, genomic version Maize B73AGP_v4). Correlation analysis between this SNP and maize ear height showed that when the mutation was C, the ear height was significantly higher than that of the T variant, as shown in the results below. Figure 1 As shown in Figure a, ear height is significantly correlated with drought resistance in maize; therefore, this invention hypothesizes that this SNP site may be related to drought resistance in maize.

[0037] Genotyping was performed on the 18 different maize inbred lines shown in Table 1, using the following method:

[0038] Using maize DNA as a template, PCR amplification was performed using Novizan's 2×Rapid Taq Master Mix. The PCR amplification reaction system consisted of: 10 μL of 2×Rapid Taq Master PCR mix, 0.5 μL each of forward and reverse primers, 1 μL of DNA template, and 8 μL of double-distilled water to a final volume of 20 μL. The reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 8 s, 56℃ annealing for 8 s, 72℃ extension for 8 s, for a total of 40 cycles; 72℃ for 5 min.

[0039] The PCR products were detected by agarose gel electrophoresis, and the results are as follows: Figure 1 As shown in Figure b, there was no significant difference in fragment size. However, sequencing analysis revealed that these 18 different maize inbred lines could be divided into two different haplotypes (T-type and C-type), as shown in the results. Figure 1 As shown in c.

[0040] The primer pairs used for the above genotyping detection are shown below:

[0041] GRF24-SNP-up: 5'-ATGTCTTTCACATGTCAATTGTCTC-3' (SEQ ID NO. 1);

[0042] GRF24-SNP-dn: 5'-TGGCAGTCAACTGTTGTGCTG-3' (SEQ ID NO. 2).

[0043] Further comparison revealed that when the amplified fragment contained the sequence shown in SEQ ID NO.3, it indicated that the maize material had high drought resistance: 5'-CCAACTTTTTATGAGCCACTAAGGA-3' (SEQ ID NO.3).

[0044] The results of haplotype typing of 18 different maize inbred lines and the data on drought resistance and yield (ear weight, grain weight) traits of maize at the adult stage under drought conditions (see...) Figure 2 Correlation analysis with Table 2 showed that haplotype C maize inbred lines had better field drought resistance, and their single ear weight and ear grain weight were significantly higher than those of type T.

[0045] Table 2. Data on drought resistance and yield (single ear weight, grain weight) of maize under drought conditions at the adult stage.

[0046] genotype Single ear weight (g) Grain weight per ear (g) T 39.7075 21.34909 C 50.75868 25.78537

[0047] The results above show that the SNP molecular markers provided by this invention are significantly correlated with yield traits (ear weight and grain weight) of maize plants under drought conditions. When the nucleotide sequence position 2-43089691 is C, the yield indicators such as ear weight and grain weight are significantly improved, indicating that these SNP molecular markers can be used to screen for drought-resistant maize materials. The SNP molecular markers of this invention can also be used for molecular-assisted breeding and identification of high-quality germplasm resources in maize, thereby accelerating the creation of drought-resistant maize materials and the breeding process of new varieties. Therefore, this invention provides a theoretical basis and technical support for marker-assisted breeding of maize for stress resistance.

[0048] 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. A SNP molecular marker associated with drought resistance in maize, characterized in that, The SNP molecular marker is located at 43089691 of chromosome 2 of the corn reference genome Maize B73 AGP_v4, and the polymorphism is T / C.

2. A primer pair for amplifying the SNP molecular marker of claim 1, characterized in that, The upstream primer comprises a nucleotide sequence as shown in SEQ ID NO. 1, and the downstream primer comprises a nucleotide sequence as shown in SEQ ID NO.

2.

3. The primer pair of claim 2 is applied in the preparation of a kit for identifying drought resistance of corn.

4. A kit for identifying drought resistance in maize, characterized in that, The kit comprises the primer pair of claim 2.

5. The kit of claim 4, wherein The kit further comprises 2x Rapidtaq Master PCR mix.

6. The SNP molecular marker of claim 1 or the primer pair of claim 2 or the kit of claim 4 is applied in any one of the following: (1) identifying drought resistance of corn; (2) identifying and screening corn varieties or lines with drought resistance; (3) corn drought resistance molecular marker assisted breeding; (4) improving corn drought resistance germplasm resources; (5) identifying and screening corn varieties or lines with high yield.

7. A method of identifying drought resistance in maize, characterized by, The method comprises the following steps: Using the genomic DNA of the corn sample to be detected as a template, the primer pair of claim 2 is used for PCR amplification to obtain an amplification product; when the amplification product contains an amplification fragment with a nucleotide sequence as shown in SEQ ID NO. 3, it is indicated that the drought resistance of the corn sample to be detected is high.

8. The method of claim 7, wherein, The reaction system of the PCR amplification is as follows: 2x Rapid taq Master PCR mix 10 μL, upstream and downstream primers each 0.5 μL, DNA template 1 μL, and double distilled water 8 μL.

9. The method of claim 7, wherein, The reaction procedure of the PCR amplification is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 8 s, 56℃ annealing for 8 s, 72℃ extension for 8 s, 40 cycles; 72℃ for 5 min.

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