Molecular markers closely linked to cold resistance traits in maize and their applications

Abstract

This invention provides a molecular marker closely linked to the cold-resistance trait in maize and its application. The molecular marker is located at 175,949,784 bp on maize chromosome 4, with a nucleotide polymorphism of T / A. The SNP molecular marker disclosed in this invention, which is significantly associated with the low-temperature germination index of maize, can be used in molecular-assisted breeding for the cold-resistance trait in maize. Furthermore, through molecular breeding improvement, screening, and cultivation of cold-resistance maize varieties, it can help expand the maize planting area and thus increase maize yield.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a molecular marker closely linked to the cold-resistance trait of maize and its application. Background Technology

[0002] Corn is my country's largest grain crop, with an annual output exceeding 260 million tons. Its planting areas are widely distributed in major producing regions such as the spring-sown corn region in Northeast China and the summer-sown corn region in the Huang-Huai-Hai Plain. However, high-latitude and high-altitude areas in the north frequently suffer from low-temperature damage, severely inhibiting seed germination: cell membrane lipid phase transitions lead to leakage, mitochondrial energy synthesis is hindered, and reactive oxygen species accumulate in large quantities, ultimately resulting in low emergence rates, weak seedling vigor, and poor uniformity, causing yield reduction and seriously threatening national food security. Traditional countermeasures, such as mulching, adjusting sowing dates, or using chemical coating agents to temporarily regulate the seed microenvironment, are costly, passive, and unsustainable. Therefore, cultivating new varieties with strong low-temperature germination ability from a genetic perspective is the most economical and sustainable fundamental way to overcome current production constraints.

[0003] In recent years, most research on the molecular mechanisms of cold tolerance in maize has focused on the seedling stage. Several transcription factors, such as COOL1, ZmDREB1, and ZmICE1, have been confirmed to participate in low-temperature signal transduction, and their overexpression can enhance the cold tolerance of maize seedlings to some extent. Seed germination, as a unique developmental stage, exhibits physiological and biochemical processes (such as storage substance mobilization, metabolic restart, and cell division and elongation activation) that are distinctly different from those of differentiated seedling tissues. Therefore, the genetic basis regulating low-temperature tolerance during germination is likely to be highly stage-specific. Currently, despite gene-level exploration, there is a severe shortage of major genes that have been cloned, functionally validated, and proven to play a dominant role at this stage, and effective genetic targets to guide the development of cold-tolerant germination varieties are still lacking. Summary of the Invention

[0004] In view of this, the present invention provides a molecular marker closely linked to the cold resistance trait of maize and its application, which can be used in molecular-assisted breeding of maize cold resistance traits, and can improve, screen and breed cold-resistant maize varieties through molecular breeding, thereby increasing maize yield.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a molecular marker located at 175949784bp on chromosome 4 of maize, with a nucleotide polymorphism of T / A.

[0006] It should be noted that, through genome-wide association analysis, this invention identified a significant single nucleotide polymorphism (SNP) site, chr4_175949784, associated with maize germination index under low-temperature stress. This site is located at 175949784 bp on chromosome 4 of the maize B73 RefGen_v5 reference genome, with a P-value of 2.74E-08. The polymorphism is T / A and is located at... Zm00001eb191140 At the -677bp promoter position of the gene (chr4: 175939332-175949107), the polymorphism of this SNP site was significantly associated with the low-temperature germination index in the tested maize materials.

[0007] The KASP molecular marker of this invention is located at 175949784bp on chromosome 4, with a base difference of T / A. Maize with base A has a strong ability to germinate at low temperatures, while maize with base T has a weaker ability to germinate at low temperatures. The polymorphism of A is the dominant trait.

[0008] Secondly, the present invention provides a primer pair combination, the primer pair combination comprising: SNP_chr4_175949784_F1, nucleotide sequence as shown in SEQ ID NO:1; specifically: 5'-GAAGGTGACCAAGTTCATGCTCGATCTCTCGTACTATGGGCTCA-3' SNP_chr4_175949784_F2, nucleotide sequence as shown in SEQ ID NO:2; specifically: 5'-GAAGGTCGGAGTCAACGGATTCGATCTCTCGTACTATGGGCTCT-3' SNP_chr4_175949784_R, nucleotide sequence as shown in SEQ ID NO:3, specifically: 5'-CCCATGCATAGACATTTTATG-3'.

[0009] Preferably, the nucleotide sequence of SNP_chr4_175949784_F1 includes a universal tag for the FAM fluorescent channel, and the nucleotide sequence of SNP_chr4_175949784_F2 includes a universal tag for the HEX fluorescent channel.

[0010] Preferably, the nucleotide sequence of the universal tag for the FAM fluorescent channel is GAAGGTGACCAAGTTCATGCT, and the universal tag for the HEX fluorescent channel is GAAGGTCGGAGTCAACGGATT.

[0011] Thirdly, the present invention provides an application of the aforementioned molecular marker in detecting the germination ability of maize under low-temperature conditions.

[0012] Fourthly, this invention provides a method for detecting the germination index of maize under low-temperature conditions. When the haplotype formed by the SNP marker at position 175949784 bp on chromosome 4 is A, maize exhibits strong germination ability under low-temperature conditions; when it is T, maize exhibits weak germination ability under low-temperature conditions. Specifically, the maize gene is sequenced, and based on the sequencing results, the genotype of the SNP molecular marker of this invention is detected. When the haplotype formed by the SNP marker at position 175949784 bp on chromosome 4 is A, maize exhibits strong germination ability under low-temperature conditions. The sequencing method includes, but is not limited to, whole-genome resequencing, targeted sequencing, or multiplex PCR sequencing.

[0013] Fifthly, this invention provides an application of the aforementioned molecular markers in maize genetic breeding and germplasm resource improvement. Specifically, genotyping of SNP markers in maize samples is performed, and maize samples with haplotype A are selected for breeding.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The SNP molecular markers disclosed in this invention that are significantly associated with the low-temperature germination index of maize can be used in molecular-assisted breeding of maize's cold-resistant traits. Furthermore, through molecular breeding improvement, cold-resistant maize varieties can be screened and cultivated, thereby increasing maize yield.

[0015] (2) This invention deeply integrates genome-wide association analysis with competitive allele-specific PCR technology to develop a molecular marker development and application system for cold-resistant traits in maize. This technology has three core advantages: site innovation, detection accuracy, and application economy, realizing a closed loop from gene mining to breeding application. It can systematically solve the problem of early spring low temperature seedling emergence in maize and provide an irreplaceable key technical tool for the rapid breeding of new cold-resistant maize varieties. Attached Figure Description

[0016] Figure 1 This is a genome-wide association analysis result of the relative germination index (RGI) of maize under low temperature stress provided in Example 1 of the present invention; Figure 2 Box plot of RGI haplotype analysis of different genotypes at locus chr4_175949784 in 322 related population inbred lines provided in Example 1 of the present invention; Figure 3 The image shows the KASP genotyping results of the chr4_175949784 marker site provided in Example 2 of this invention in 40 maize test materials.

[0017] in, Figure 1In the diagram, Figure A is the Manhattan plot and Figure B is the QQ-plot. Figure 3 In the diagram, the horizontal axis represents the TT genotype, indicated by a blue square; the vertical axis represents the AA genotype, indicated by a red square; and the black square represents the negative control. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0019] Example 1: GWAS and haplotype analysis related to low-temperature germination in maize 1.1 Phenotypic Survey This study used a natural variation-associated population covering superior backbone inbred lines of major maize varieties in Northeast China, totaling 322 accessions. For each inbred line, 60 normal seeds without damage or mold were selected, disinfected with 1% sodium hypochlorite for 15 min, rinsed 5 times with deionized water, and air-dried. The seeds were then placed embryo-side up in sterile petri dishes, which were then wrapped with aluminum foil to prevent excessive moisture loss. Germination at room temperature was carried out in a 26℃ incubator in the dark. Germination counts were started after 12 hours and repeated every 12 hours until the germination rate stabilized (generally 72 hours). Germination at low temperature was carried out in a 6℃ incubator in the dark. Germination counts were started after 2 days and repeated every 2 days until the germination rate stabilized (generally ending on day 14). The germination index (GI) was calculated under both room temperature and low temperature conditions. The relative germination index (RGI) (the ratio of the 6℃ germination index to the 26℃ germination index) was used as the phenotypic value for GWAS analysis.

[0020] 1.2 GWAS analysis and identification of significant SNP sites 322 maize inbred lines from related populations were resequencing by a sequencing company. Using the selection criteria of minimum allele frequency (MAF) > 0.05 and deletion rate < 20%, only biallelic loci were retained, resulting in 6.25 million high-quality SNP genotypes. GWAS analysis of the relative germination index (RGI) for cold hardiness was performed using the MLM model in TASSEL 5.0 software. 10 (P-value)=5 was used as a threshold to determine significantly associated SNP sites. Figure 1 The results showed that the SNP locus chr4_175949784 on chromosome 4 was significantly associated with the germination phenotype of maize under low temperature conditions, with a P-value of 2.74E-08 and a nucleic acid polymorphism of T / A. Information on this SNP locus is detailed in Table 1.

[0021] Table 1. Information on SNP sites significantly associated with maize low-temperature germination index.

[0022] 1.3 Haplotype Analysis Haplotype analysis was performed by combining SNP loci with the low-temperature germination publication types of 322 tested maize inbred lines. Figure 2 Among them, SNP locus variation genotyping was divided into two types. Specifically, based on the T / A base difference, 292 inbred lines with genotype TT had poor germination rate at low temperatures, with an average relative germination index of 0.015. 30 inbred lines with genotype AA had higher germination rate at low temperatures, with a relative germination index of 0.065. The difference in genotypes between the two haplotypes and low-temperature germination was 1.8E-12, reaching a significant level (P<0.05). The relative germination index of the AA genotype was significantly higher than that of the TT genotype maize inbred lines.

[0023] Example 2: Development and application of KASP molecular markers related to low-temperature germination in maize 2.1 Test materials Twenty inbred lines from related populations with significant differences in relative germination index (RGI) under low-temperature conditions were selected, and the accuracy was verified using the developed KASP molecular marker. The specific materials used are shown in Table 2, including 10 materials with strong low-temperature germination ability and 10 materials with weak low-temperature germination ability.

[0024] Table 2. Low-temperature relative germination index (GRI) data and genotypes of 20 maize inbred lines.

[0025] 2.2 Development of KASP Tags Based on the SNP locus information obtained above, a variation was found at 175,949,784 bp on chromosome 4 of the reference genome B73 RefGen_v5, with a polymorphism of T / A. Flanking sequences of 150 bp before and after this locus were extracted, and primers were designed. This KASP molecular marker consists of three primers: two specific forward primers (SEQ ID NO.1 and SEQ ID NO.2) and one universal reverse primer (SEQ ID NO.3). The primers were synthesized by Beijing Liuhe BGI Genomics Co., Ltd., and the primer sequences are as follows: SNP_chr4_175949784_F1 (SEQ ID NO.1) 5'-GAAGGTGACCAAGTTCATGCTCGATCTCTCGTACTATGGGCTCA-3' SNP_chr4_175949784_F2 (SEQ ID NO.2) 5'-GAAGGTCGGAGTCAACGGATTCGATCTCTCGTACTATGGGCTCT-3' SNP_chr4_175949784_R (SEQ ID NO.3)) 5'-CCCATGCATAGACATTTTATG-3' Among them, GAAGGTGACCAAGTTCATGCT is a universal tag for FAM fluorescent adapter sequences; GAAGGTCGGAGTCAACGGATT is a universal tag for HEX fluorescent adapter sequences.

[0026] 2.3 DNA Extraction (1) Take about 0.1g of fresh corn material and put it into a 1.5mL centrifuge tube. Add 2 steel balls and put it into liquid nitrogen for quick freezing. After freezing, shake it in a grinder for 2 minutes to destroy the cell wall and cell membrane and release the nucleoprotein.

[0027] (2) Use 400 μL CTAB extraction buffer to lyse the sample at 65°C to separate the nucleoprotein from the lysed cell fluid (this process takes about 30 minutes, during which the sample is inverted 3-4 times).

[0028] (3) After cooling to room temperature, add 400 μL of chloroform:isoamyl alcohol (24:1), mix by inverting, centrifuge to remove denatured proteins, polysaccharides and phenols and other impurities, and retain the supernatant.

[0029] (4) Add 250 μL of isopropanol to the supernatant, mix gently, and place in a -30°C freezer to precipitate DNA (this process can be precipitated for 30 min or longer without affecting the quality of DNA extraction).

[0030] (5) Centrifuge at 10,000 rpm for 10-15 min, discard the supernatant, wash the precipitate twice with 70% ethanol, and the white precipitate is the genomic DNA.

[0031] (6) After the ethanol has completely evaporated, add 300 μL of deionized water to dissolve the DNA. The DNA can be stored for a long time in a -20°C refrigerator.

[0032] 2.4 KASP Classification The DNA concentration of the sample to be tested was diluted to 30 ng / L, and each sample was tested in duplicate for KASP detection. The KASP molecular marker PCR amplification reaction system was as follows: 5 μL KASP 2X PCR mix, 0.5 μL primer premix (mixing SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 primers at a concentration of 10 μM in a volume ratio of 2:2:5), 1.5 μL template DNA, and ddH2O to a final volume of 10 μL.

[0033] The PCR amplification program was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 61℃ annealing extension for 60 s, 10 cycles, with the annealing extension temperature decreasing by 0.6℃ each time; the second amplification reaction was 95℃ denaturation for 15 s, 55℃ annealing extension for 60 s, 30 cycles.

[0034] After the reaction, the fluorescence data of the amplified PCR products were read using the QuantStudio™ Design Analysis Software v1.5.2 real-time quantitative PCR instrument. The fluorescence scanning results can be converted into graphs. Figure 3 According to the color classification of the genotyping data read by the quantitative real-time PCR instrument, samples aggregated near the X-axis represent genotype TT linked to HEX fluorescent tag sequences, displayed as blue squares; samples aggregated near the Y-axis represent genotype AA linked to FAM fluorescent tag sequences, displayed as red squares; black squares represent negative controls. Comparison of the genotyping results from the quantitative real-time PCR instrument with the resequencing results of the chr4_175949784 locus showed that the locus variation type detected by the KASP molecular marker was consistent with the resequencing variation results, indicating that the KASP marker for this SNP locus was successfully developed.

[0035] As expected, the KASP molecular marker of this invention can effectively identify the germination phenotype of maize under low temperature stress and can be used for the prediction and screening of cold-resistant maize varieties.

[0036] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0037] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A molecular marker, characterized in that, Located at 175949784bp on chromosome 4 of maize, the nucleotide polymorphism is T / A.

2. A primer pair combination, characterized in that, The primer pair combinations include: SNP_chr4_175949784_F1, nucleotide sequence as shown in SEQ ID NO:1; SNP_chr4_175949784_F2, nucleotide sequence as shown in SEQ ID NO:2; SNP_chr4_175949784_R, nucleotide sequence as shown in SEQ ID NO:

3.

3. The primer pair combination according to claim 2, characterized in that, The nucleotide sequence of SNP_chr4_175949784_F1 includes a universal tag for the FAM fluorescent channel, and the nucleotide sequence of SNP_chr4_175949784_F2 includes a universal tag for the HEX fluorescent channel.

4. The primer pair combination according to claim 3, characterized in that, The universal tag nucleotide sequence for the FAM fluorescent channel is GAAGGTGACCAAGTTCATGCT, and the universal tag for the HEX fluorescent channel is GAAGGTCGGAGTCAACGGATT.

5. The application of the molecular marker according to claim 1 in detecting the germination ability of maize under low temperature conditions.

6. A method for detecting the germination index of maize under low-temperature conditions, characterized in that, When the haplotype formed by the SNP marker at position 175949784bp on chromosome 4 is A, maize has a strong germination ability under low temperature conditions; when it is T, maize has a weak germination ability under low temperature conditions.

7. The application of the molecular markers according to claim 1 in maize genetic breeding and germplasm resource improvement.

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