InDel markers for identifying trace element Cd in maize kernels and their applications

Abstract

This invention discloses InDel marker primers for identifying the trace element Cd in maize kernels and their applications. The primer sequences are: F: 5'-CCAGTTCTGGAAATTCCTCCT-3'; R: 5'-ATGGTGCCATGTGTCTGAAA-3'. The inventors discovered that the genotype at position 149203081 bp on maize chromosome 2 is highly correlated with the Cd content of maize inbred lines. Specifically, the InDel genotype is a single-banded type A, indicating high Cd content in the kernels, while the InDel genotype is a heterozygous type H, indicating low Cd content. This genotype can be used as an InDel marker to detect the Cd content in maize kernels. Using this InDel marker, maize inbred lines with high trace element levels can be bred.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to InDel-labeled primers for identifying trace element Cd in maize kernels and their applications. Background Technology

[0002] Maize (Zea mays L.) is a major food crop, containing various trace elements and chemical substances beneficial to the human body, such as selenium. It has a strong ability to accumulate cadmium (Cd), but the Cd content in the kernels is relatively low. Both excessively high and low levels of Cd can affect the taste and quality of maize. Considering both the nutritional value and safety risks of maize kernels, to avoid Cd entering the human body through the food chain, it is necessary to sacrifice its biological characteristic of accumulating trace elements. Molecular marker verification can serve as a method to identify maize inbred lines with high or low Cd content and to prioritize the selection of inbred lines with suitable Cd content in agricultural production. Therefore, elucidating the genetic mechanism of Cd traits in maize kernels is of great significance for improving the nutritional quality and food safety of maize. Summary of the Invention

[0003] The inventors have discovered that the genotype located at position 149203081bp on chromosome 2 of maize is highly correlated with the Cd content in maize kernels. When the genotype at position 149203081bp on chromosome 2 of maize is the single-banded type A, the Cd content in maize kernels is high; when the genotype at position 149203081bp on chromosome 2 of maize is the heterozygous type H, the Cd content in maize kernels is low.

[0004] Therefore, the first objective of this invention is to provide an InDel-labeled primer for identifying the trace element Cd in maize kernels, wherein the primer sequence is as follows:

[0005] F: 5'-CCAGTTCTGGAAATTCCTCCT-3' (SEQ ID NO. 1);

[0006] R: 5'-ATGGTGCCATGTGTCTGAAA-3' (SEQ ID NO. 2).

[0007] A second objective of this invention is to provide a kit for identifying the Cd content of corn kernels, which contains the primers described above.

[0008] Preferably, the kit further includes reagents for extracting maize genomic DNA to be tested.

[0009] A third objective of this invention is to provide the application of the aforementioned primers or kits in maize germplasm improvement.

[0010] A fourth objective of this invention is to provide the application of the aforementioned primers or kits in identifying maize inbred lines with high or low Cd content.

[0011] Preferably, in the aforementioned application, primers are used for PCR amplification. When the genotype of the amplified product is a single-banded type A, the Cd content of the trace element in the corn kernels is high; when the genotype of the amplified product is a heterozygous type H, the Cd content of the trace element in the corn kernels is low.

[0012] The fifth objective of this invention is to provide a method for identifying the Cd content in corn kernels, comprising the following steps:

[0013] (1) Extract genomic DNA from the maize to be tested;

[0014] (2) Using the extracted DNA as a template, perform PCR amplification using the primers described above;

[0015] (3) Perform electrophoresis on the PCR amplification products and identify the Cd content of corn kernels based on the electrophoresis results.

[0016] Preferably, in the method, when the genotype of the amplified product is a single-band type A, the Cd content of the corn kernel is high; when the genotype of the amplified product is a heterozygous type H, the Cd content of the corn kernel is low.

[0017] The sixth objective of this invention is to provide the application of the above-described method in maize germplasm improvement.

[0018] The seventh objective of this invention is to provide the application of the above-described method in identifying maize inbred lines with high or low Cd content.

[0019] The beneficial effects of this invention are as follows: The inventors discovered that the genotype at position 149203081 bp on maize chromosome 2 is highly correlated with the Cd content of maize inbred lines. Specifically, the InDel genotype is a single-banded type A, indicating high Cd content in maize kernels, while the InDel genotype is a heterozygous type H, indicating low Cd content. This genotype can be used as an InDel marker to detect the Cd content in maize kernels. This InDel marker can be used to breed maize inbred lines with high Cd content. Attached Figure Description

[0020] Figure 1 This is a Manhattan plot of the GWAS analysis results. The arrow points to the SNP at position 149203081 bp on chromosome 2.

[0021] Figure 2This shows the amplification results of primer Chr2Cd in natural population samples. The two bands in one lane represent different samples with low levels of trace element Cd, while the single band in another lane represents different samples with high levels of trace element Cd. Detailed Implementation

[0022] The following are specific implementation examples of the present invention. It should be noted that these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Modifications and substitutions to the details and form of the implementation schemes made within the scope and spirit of the present invention all fall within the protection scope of the present invention.

[0023] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents.

[0024] Example 1

[0025] 1. Research Methods

[0026] (1) Phenotypic determination

[0027] This study used a natural population of 163 inbred lines, planted in Wengyuan County, Shaoguan City, Guangdong Province in the second half of 2020. The planted materials were self-pollinated. After maturity, seeds from different inbred lines in the natural population were selected, dried at 80℃ for 3 days, and then ground into powder, with 5g of each sample weighed. The samples were digested in Anton Paar Multiwave PRO microwave with 65% nitric acid (mass fraction) at a temperature gradient from 120℃ to 180℃ for 45 minutes, followed by cooling for 30 minutes. After dilution in deionized water and bringing the volume to 10 mL, the concentrations of 12 elements were measured by inductively coupled plasma mass spectrometry (ICP-MS). The average of three replicates for each material was used as the phenotypic value of the trace elements in the seeds of the inbred lines.

[0028] (2) Genome-wide association analysis (GWAS)

[0029] Genome-wide association analysis (GWA) was performed using micronutrient-related phenotypic data of maize kernels, combined with 12,284,796 SNPs covering the entire maize genome, employing a mixed linear model (MLM). A significance level of ≤1 / 198910 (P≤1×10⁻⁶) was selected for the association analysis. -6 This value serves as the significance threshold for the GWAS results.

[0030] (3) Genotyping

[0031] The aforementioned populations separating micronutrient content in maize grains were used as test populations for developing molecular markers for micronutrient content in maize grains. Simultaneously, extreme populations with significant differences in micronutrient content traits from previous GWAS mapping experiments were used as experimental materials to verify the effectiveness of the molecular markers. Based on the results of the previous GWAS mapping experiments, highly significant SNP sites and SNP regions with concentrated significant sites were selected. Furthermore, based on the InDel location reported by Mo17 on the B73v4 reference genome, InDel molecular markers were designed and screened and validated in natural populations.

[0032] 2. Research Results

[0033] (1) Phenotypic distribution of Cd trace element in maize kernels in related populations

[0034] The study of this invention measured the Cd content of 163 maize inbred lines, obtained the Cd content index, and performed descriptive statistical analysis on the measured Cd content data, as shown in Table 1.

[0035] Table 1. Descriptive statistics of Cd content in maize kernels from a population.

[0036]

[0037] (2) Genome-wide association analysis

[0038] Combining tens of millions of SNP markers and phenotypic data on Cd content in grains, the inventors used genome-wide association analysis to identify an SNP marker located at position 149203081 bp on maize chromosome 2 that was significantly associated with Cd content in maize inbred lines. Figure 1The polymorphic site chr2:149203081bp linked to this site was selected, and primers Chr2Cd F: 5'-CCAGTTCTGGAAATTCCTCCT-3' (SEQ ID NO.1) and R: 5'-ATGGTGCCATGTGTCTGAAA-3' (SEQ ID NO.2) were designed to amplify this site, and the correlation between this polymorphic site and the Cd content in grains was detected. Grains with the Cd trait selected from natural populations were used as templates for PCR amplification using genomic DNA from these samples to verify whether the amplification product of this marker in natural populations is polymorphic. PCR amplification conditions: PCR reaction parameters were set as follows: 94℃ pre-denaturation for 30s, 98℃ denaturation for 10s, annealing temperature of 55℃ for 30s, extension at 72℃ for 30s, for a total of 35 cycles, with a final extension at 72℃ for 2min. The PCR reaction system consisted of 5 μL of 2×Pro Taq Mix, 0.5 μL of primer-F (10 μM), 0.5 μL of primer-R (10 μM), 1 μL of DNA template (50 ng / μL), 3 μL of ddH2O, and a total volume of 10 μL.

[0039] Electrophoresis results of the amplification products showed that in the amplification results of 7 materials with low Cd content, the gene banding pattern was heterozygous H type (two bands); in the amplification results of 73 materials with high Cd content, the gene banding pattern was single band A type (one band). Figure 2 At a confidence level of 0.01, the electrophoresis results were statistically analyzed and subjected to analysis of variance (Table 2). The results showed a significant difference between the two groups of data. Therefore, it is believed that this primer is still effective in identifying the Cd content of grains based on natural maize populations, and can provide some theoretical support for guiding grain nutritional value and safe breeding.

[0040] Table 2. Analysis of variance of data from two groups of natural populations.

[0041]

[0042] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of an InDel marker primer related to the trace element Cd in corn kernels or a kit containing the primer in identifying corn inbred lines with high or low trace element Cd content, characterized in that, The primer sequence is: F: 5'-CCAGTTCTGGAAATTCCTCCT-3'; R: 5'-ATGGTGCCATGTGTCTGAAA-3'.

2. Use according to claim 1, characterized in that, When the genotype of the amplification product is single band type A, the content of trace element Cd in the corn kernel is high; when the genotype of the amplification product is hybrid band type H, the content of trace element Cd in the corn kernel is low.

3. A method for identifying the content of trace element Cd in corn kernels, characterized in that, The method comprises the following steps: (1) extracting the genomic DNA of the corn to be detected; (2) taking the extracted DNA as a template, and performing PCR amplification by using the InDel marker primer for identifying the corn kernel trace element Cd-related; the primer sequence is: F: 5'-CCAGTTCTGGAAATTCCTCCT-3'; R: 5'-ATGGTGCCATGTGTCTGAAA-3'; (3) performing electrophoresis on the PCR amplification product, and identifying the content of the corn kernel trace element Cd according to the electrophoresis result.

4. The method of claim 3, wherein, When the genotype of the amplification product is single band type A, the content of trace element Cd in the corn kernel is high; when the genotype of the amplification product is hybrid band type H, the content of trace element Cd in the corn kernel is low.

5. The method of claim 3 is applied in the improvement of corn germplasm.

6. The method of claim 3 is applied in identifying the corn inbred lines with high or low content of trace element Cd.

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