A molecular marker for detecting zinc content of wheat kernels and application thereof

Abstract

The application belongs to the technical field of molecular marker detection, and particularly relates to a molecular marker for detecting the zinc content of wheat kernels and application thereof. TaNAS-6B The g.723 G>A site in the interior is, for example, the 723th nucleotide in SEQ ID NO:1, and the nucleotide type is G or A. The application further provides a method for identifying or assisting in identifying the zinc content trait of wheat kernels, which comprises detecting the genotype of the g.723 G>A site in the wheat to be detected, and the wheat to be detected with the genotype AA is high-zinc wheat, and the zinc content of the kernels is significantly higher than that of the wheat to be detected with the genotype GG. The molecular marker provided by the application can be used for detecting the genotype of TaNAS-6B , and is applied to the improvement breeding of the zinc content of wheat kernels, and can improve the efficiency of selection breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker detection technology, specifically relating to a molecular marker for detecting zinc content in wheat grains and its application. Background Technology

[0002] Zinc is one of the 15 essential trace elements for the human body. Zinc deficiency can lead to stunted growth, skin diseases, nervous system disorders, and various immune-related illnesses. The human body cannot synthesize zinc and must obtain it from external sources through diet. Grains are the primary source of trace elements for humans. Wheat is one of the world's major food crops. If zinc-enriched wheat varieties can be developed through biofortification breeding to increase the zinc content in wheat grains, zinc deficiency can be largely eliminated, thus safeguarding public health.

[0003] Zinc content in wheat grains is significantly influenced by environmental factors, and its measurement requires large seed sample sizes. Therefore, effective measurement and screening are not feasible in early generations of breeding, resulting in low accuracy and efficiency when using traditional breeding methods for zinc content selection. In recent years, the rapid development of wheat functional genomics and molecular markers has provided opportunities for the genetic improvement of zinc content in wheat grains. Molecular markers can effectively identify genetic loci related to zinc content in wheat grains at the genetic level, and using marker-assisted selection strategies for genetic improvement of zinc content in wheat grains can greatly improve breeding selection efficiency, thereby shortening the breeding cycle. Summary of the Invention

[0004] This invention provides a molecular marker for detecting zinc content in wheat grains. This marker can be used to identify or screen wheat grain zinc content, providing more options for wheat quality improvement breeding.

[0005] In a first aspect, the present invention provides a product for detecting the g.723G>A site within the TaNAS-6B gene, the product comprising:

[0006] (1) A primer composition for detecting the genotype of the g.723G>A site within the TaNAS-6B gene, wherein the g.723G>A site is a SNP site within the TaNAS-6B gene in the wheat genome, specifically the 723rd nucleotide of SEQ ID NO:1, and its nucleotide type is G or A; the primer composition comprises:

[0007] SNP723-F: 5'-CTCAAAGCCGGACGTCAGAG-3';

[0008] SNP723-R: 5'-TCGTGGACCAACAGAGAACC-3';

[0009] (2) A reagent or kit comprising the primer combination described in (1). The reagent or kit may also include the restriction endonuclease Hinf I or sequencing reagents.

[0010] In a second aspect, the present invention provides the application of the above-described product in any of the following:

[0011] (1) Identify the allele genotype of the g.723G>A site within the wheat TaNAS-6B gene;

[0012] (2) To identify or assist in the identification of zinc content in wheat grains;

[0013] (3) Screening or assisted screening of wheat plants, strains, varieties or strains with high zinc content in wheat grains;

[0014] (4) Prepare products for identifying or assisting in the identification or comparison of the zinc content of wheat grains to be tested;

[0015] (5) Marker-assisted selection breeding of wheat.

[0016] A third aspect of this invention provides a method for identifying the allele genotype at the g.723G>A site within the TaNAS-6B gene, specifically comprising the following steps: using the genomic DNA of the wheat variety / line to be tested as a template, performing PCR amplification using the primer pair in the above product, wherein the amplification product is 1488 bp in length; then digesting the above PCR amplification product with the restriction endonuclease Hinf I, followed by agarose gel electrophoresis, and interpreting the band types; after enzyme digestion, the amplification product can be distinguished into two band types, 946 bp and 542 bp, which have the GG allele genotype; after enzyme digestion, only one band type, 1488 bp, has the AA allele genotype; or the PCR amplification product can be sequenced to obtain the allele genotype at the g.723G>A site within the TaNAS-6B gene.

[0017] This invention can identify or assist in identifying the zinc content trait of wheat grains based on the genotype of the wheat to be tested. Therefore, in a fourth aspect, this invention provides a method for identifying the zinc content trait of wheat grains, comprising: obtaining the genotype of the wheat to be tested using the method described above for identifying the allele genotype of the g.723G>A locus within the TaNAS-6B gene; and then identifying or assisting in identifying the zinc content trait of the wheat grains based on the genotype, wherein the zinc content of the wheat grains of the wheat with the genotype AA at the g.723G>A locus is higher than that of the wheat grains of the wheat with the genotype GG at the g.723G>A locus; the genotype GG represents a homozygous type of nucleotide G at the g.723G>A locus in the wheat genome; and the genotype AA represents a homozygous type of nucleotide A at the g.723G>A locus in the wheat genome.

[0018] In a fifth aspect, this invention provides a method for breeding high-quality new wheat varieties / lines, comprising the following steps:

[0019] (1) The above method was used to detect the allele genotype of the g.723G>A site within the TaNAS-6B gene in candidate wheat breeding materials;

[0020] (2) Select wheat breeding materials with AA alleles as the male / female parent for subsequent breeding.

[0021] Compared with the prior art, the main beneficial technical effects of the present invention are as follows:

[0022] 1. This invention analyzed the cloned wheat grain zinc content regulating gene TaNAS-6B and found that the g.723G>A site within it is significantly associated with the wheat grain zinc content trait. Based on this, this invention developed the g.723G>A site into a dCAPS marker. Studies have demonstrated that the dCAPS marker described in this invention can be used to detect the TaNAS-6B genotype, with a simple method and high accuracy.

[0023] 2. The molecular markers developed in this invention can be applied to marker-assisted selection breeding of wheat to achieve early screening of zinc content traits in wheat grains, which can greatly improve the efficiency of selection breeding. Attached Figure Description

[0024] Figure 1 This is a haplotype analysis diagram of the TaNAS-6B gene in Example 1 of the present invention.

[0025] Figure 2 This is a comparison chart of the zinc content in grains among the four haplotypes of the TaNAS-6B gene in Example 1 of the present invention.

[0026] Figure 3 This is a diagram showing the enzyme digestion results of the amplification products of the SNP723-F and SNP723-R primer pairs in Example 2 of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0028] Unless otherwise specified, all instruments and equipment used in the following embodiments are conventional instruments and equipment; all reagents, carriers, and other experimental materials used are commercially available conventional products; and all experimental methods and detection methods used are conventional methods. Quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged. Primer synthesis and sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0029] The wheat material involved in the examples:

[0030] A natural population of 442 wheat varieties / lines was used for haplotype analysis of the TaNAS-6B gene, which regulates zinc content in wheat grains, and for association analysis between the alleles of the g.723G>A locus within the TaNAS-6B gene and the phenotype of zinc content in grains.

[0031] Example 1: Discovery of SNPs closely linked to the wheat grain zinc content trait within the TaNAS-6B gene regulating wheat grain zinc content.

[0032] 1. Group planting

[0033] Forty-two wheat varieties / lines used for TaNAS-6B gene haplotype analysis and grain zinc content phenotypic determination were planted at the Yuanyang Research Base of Henan Agricultural University during the 2023-2024 growing season. A completely randomized block design was adopted, with each planting plot 2m long and 1.5m wide, containing 12 rows. Each variety was planted in two rows, with single-seed sowing. The plant spacing between rows and within rows was 20cm and 10cm, respectively. The experimental fields were managed according to standards, and there were no droughts, lodging, or pests and diseases during the entire growing season. After harvest, the grains were threshed, dried, and stored uniformly.

[0034] 2. Determination of zinc content in grains

[0035] The determination was performed using flame atomic absorption spectrophotometry. Wheat grains were placed in kraft paper bags, dried at 65℃ to constant weight, pulverized, and stored in plastic resealable bags. 1.0 g of the pulverized wheat grain sample was accurately weighed into a porcelain crucible, carbonized, and then transferred to a muffle furnace for ashing at 550℃ for 6 hours until the ash was nearly white. After cooling to room temperature, 5 mL of nitric acid solution (1:1 volume ratio) was added, and the mixture was allowed to stand until the ash dissolved. The solution was then filtered through filter paper into a 50 mL volumetric flask, and ddH₂O was added to bring the volume to 50 mL. Measurements were performed using a Hitachi ZA-3000 flame atomic absorption spectrophotometer, with a blank control included. Three technical replicates were performed. The zinc content in the wheat grains was calculated based on the standard curve. The average of the three measurements was taken as the final value. The measurement results are shown in Table 1.

[0036] Preparation of zinc standard curve: Add zinc standard solution to seven 50mL volumetric flasks, add ddH2O to make up to 50mL, so that the final concentrations are 0, 0.2, 0.4, 0.6, 0.8 and 1μg / mL, respectively. Measure the absorbance of each solution, and then prepare the zinc standard curve.

[0037] Table 1. Mean grain zinc content and genotypes of the g.723G>A locus in 442 wheat varieties / lines.

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] 3. Haplotype analysis of TaNAS-6B gene, a gene regulating zinc content in wheat grains

[0044] Leaves from 442 wheat varieties / lines were sampled during the seedling stage. Genomic DNA was extracted, and the coding region of the TaNAS-6B gene was amplified. The amplified product was then sequenced.

[0045] The specific steps are as follows:

[0046] ① DNA extraction: DNA was extracted from 442 wheat varieties / lines at the seedling stage using the SLS method.

[0047] The specific implementation steps are as follows:

[0048] (1) Take an appropriate amount of wheat leaves and put them into a 2ml centrifuge tube containing steel balls. Quickly freeze the tube in liquid nitrogen and then grind it with a grinder. Add 800μL SLS and shake for 20min to mix it thoroughly. The SLS contains 288mM NaCl, 200mM Tris-HCl, 25mM EDTA, and 0.5% SDS.

[0049] (2) Then add an equal volume of the three-in-one solution (phenol:chloroform:isoamyl alcohol = 25:24:1);

[0050] (3) Shake for 10 minutes to mix thoroughly;

[0051] (4) Centrifuge at 12,000 rpm for 15 min, transfer the supernatant to another new 2 mL centrifuge tube, add an equal volume of pre-cooled isopropanol, and let stand for 10 min to precipitate;

[0052] (5) Centrifuge at 12,000 rpm for 15 min, discard the supernatant, add 0.5 mL of 75% (volume percentage) ethanol aqueous solution, and let stand for 5 min.

[0053] (6) Centrifuge at 12,000 for 5 min, discard the supernatant, vacuum dry, add 100 μL TE to dissolve the precipitate, and obtain wheat leaf genomic DNA.

[0054] ②Amplification and sequence analysis of the TaNAS-6B gene coding region

[0055] PCR primers were designed, with TaNAS-6B-F and TaNAS-6B-R positioned at the start codon ATG and stop codon TAG, respectively. Specific details are as follows:

[0056] TaNAS-6B-F: 5'-ATGGAGGTGGAGTCTAGC-3';

[0057] TaNAS-6B-R: 5'-TTAGAAGGCCACTTCCGCGT-3'.

[0058] Using DNA from 442 wheat varieties / lines as templates, PCR amplification was performed using primer pairs consisting of TaNAS-6B-F and TaNAS-6B-R. The PCR amplification system is shown in Table 2, and the PCR amplification program is shown in Table 3.

[0059] Table 2. PCR amplification system of the TaNAS-6B gene using primer pairs composed of TaNAS-6B-F and TaNAS-6B-R.

[0060] Components volume 2×Phanta buffer 25μL dNTP 1μL Phanta Enzyme 1μL Primer F 2μL Primer R 2μL DNA 2μL <![CDATA[ddH2O]]> To 50μL

[0061] Table 3. PCR amplification program for the TaNAS-6B gene using primer pairs composed of TaNAS-6B-F and TaNAS-6B-R.

[0062]

[0063] The PCR products were subjected to 1% agarose gel electrophoresis. After the target band was detected, the samples were sent to Shanghai Sangon Biotech for sequencing.

[0064] The sequencing results were analyzed, and the results are as follows: Figure 1As shown, among the 442 wheat varieties / lines tested, there are a total of 9 SNP sites in the coding region of the TaNAS-6B gene, located at positions 92bp, 384bp, 390bp, 442bp, 559bp, 648bp, 684bp, 723bp, and 732bp after the start codon ATG. Based on these 9 SNP sites, the TaNAS-6B gene can be divided into four haplotypes, named Hap1, Hap2, Hap3, and Hap4.

[0065] The four haplotypes of TaNAS-6B were analyzed in conjunction with the phenotypic values ​​of grain zinc content. The results are as follows: Figure 2 As shown, wheat varieties / lines with the Hap2 type have significantly higher grain zinc content than those with the other three haplotypes. Further analysis revealed that the variant site g.723G>A, located at 723 bp after the start codon ATG, is a specific site that distinguishes Hap3 from the other three haplotypes, and this site is closely linked to grain zinc content.

[0066] Example 2: Development of dCAPS markers at the g.723G>A site within the TaNAS-6B gene

[0067] Analysis of the sequence containing the g.723G>A site within the TaNAS-6B gene revealed that this site is located on the recognition sequence of the restriction endonuclease Hinf I. When the genotype at this site is GG, it cannot be recognized and digested by Hinf I, while it can be recognized by Hinf I when the genotype is AA. Based on this, the dCAPS marker was developed. First, specific primer pairs SNP723-F and SNP723-R were designed based on the Chinese spring reference genome sequence to amplify the DNA fragment containing the g.723G>A site. The amplified fragment length was 1488 bp, and the amplified product sequence is shown in SEQ ID NO:2. The nucleotide sequences of the primer pairs are shown below:

[0068] SNP723-F: 5'-CTCAAAGCCGGACGTCAGAG-3';

[0069] SNP723-R: 5'-TCGTGGACCAACAGAGAACC-3'.

[0070] DNA from a subset of wheat varieties / lines was randomly selected from 442 wheat varieties / lines and amplified using the primer pairs described above. The PCR amplification system is shown in Table 4, and the PCR amplification procedure is shown in Table 5.

[0071] Table 4. PCR amplification system of the primer pair composed of SNP723-F and SNP723-R for the DNA fragment containing g.723G>A

[0072] Components volume 10×Taqplus buffer 3μL dNTP 1μL Taq plus DNA Polymerase 1μL Primer F 1μL Primer R 1μL DNA 2μL <![CDATA[ddH2O]]> To 30μL

[0073] Table 5. PCR amplification program for the DNA fragment containing g.723G>A using primer pairs composed of SNP723-F and SNP723-R.

[0074]

[0075] Take 20 μL of the amplified product and digest it with the restriction endonuclease Hinf I at 37 °C. The digestion reaction system is shown in Table 6.

[0076] Table 6. Enzyme digestion reaction system for SNP723-F and SNP723-R primer pair amplification products

[0077] Components volume PCR products 20μL 10×buffer 3μL Hinf I 1μL <![CDATA[ddH2O]]> To 30μL

[0078] The enzyme digestion products were subjected to 1% agarose gel electrophoresis to determine whether the DNA fragments amplified using SNP723-F and SNP723-R primer pairs could be digested by the enzymes. The results are as follows: Figure 3 As shown, the DNA fragment of wheat lines with the GG genotype at the g.723G>A locus could be successfully cleaved into two fragments of length 946 bp and 542 bp, respectively, while the DNA fragment of wheat lines with the AA genotype at the g.723G>A locus could not be cleaved. This result indicates that the dCAPS marker can be used to determine the allele genotype at the g.723G>A locus.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A detection method TaNAS-6B The application of a product containing the g.723 G>A site within the gene in any of the following is characterized by: (1) Identification of zinc content in wheat grains; (2) Screen wheat with high zinc content in wheat grains; (3) Prepare a product for identifying the zinc content of wheat grains to be tested; Among them, the zinc content of the tested wheat with genotype AA was higher than that of the tested wheat with genotype GG; the product includes: Used for detection TaNAS-6B Primer composition for the g.723 G>A site genotype within the wheat genome, or a reagent or kit containing said primer composition, wherein the g.723 G>A site is located in the wheat genome. TaNAS-6B A single SNP site within the gene, specifically nucleotide 723 of SEQ ID NO:1, whose nucleotide type is G or A; the primer composition comprises: SNP723-F :5’- CTCAAAGCCGGACGTCAGAG-3’; SNP723-R :5’- TCGTGGACCAACAGAGAACC-3’。 2. A method for identifying the zinc content trait in wheat grains, characterized in that, include: Using identification TaNAS-6B The genotype of the wheat to be tested was obtained by using the method of allele genotyping at the g.723 G>A locus within the gene, and then the zinc content trait of the wheat grain was identified based on the genotype; the zinc content of the wheat grains of the wheat with the genotype AA at the g.723 G>A locus was higher than that of the wheat grains with the genotype GG at the g.723 G>A locus. Identification TaNAS-6B The method for determining the allele genotype of the g.723 G>A site within a gene includes the following steps: using the genomic DNA of the wheat to be tested as a template, performing PCR amplification with the primer composition in the product described in claim 1; Then, restriction endonucleases were used. Hinf The PCR amplification products were digested with enzymes, followed by agarose gel electrophoresis to determine the band types. After enzyme digestion, the amplification products could be distinguished into two band types: 946 bp and 542 bp, indicating the AA allele; the amplification products with only one band type (1488 bp) indicated the GG allele. Alternatively, the PCR amplification products could be sequenced to obtain... TaNAS-6B Allele at the g.723 G>A locus within the gene.

3. A new method for breeding high-quality wheat, characterized in that, Includes the following steps: (1) Detection of candidate wheat breeding materials TaNAS-6B Alleles at the g.723 G>A locus within the gene; identification TaNAS-6B The method for determining the allele genotype of the g.723 G>A site within a gene includes the following steps: using the genomic DNA of the wheat to be tested as a template, performing PCR amplification with the primer composition in the product described in claim 1; Then, restriction endonucleases were used. Hinf The PCR amplification products were digested with enzymes, followed by agarose gel electrophoresis to determine the band types. After enzyme digestion, the amplification products could be distinguished into two band types: 946 bp and 542 bp, indicating the AA allele; the amplification products with only one band type (1488 bp) indicated the GG allele. Alternatively, the PCR amplification products could be sequenced to obtain... TaNAS-6B Allele genotype at the g.723 G>A locus within the gene; (2) Select wheat breeding materials with the AA allele type as the male / female parent for subsequent breeding; The zinc content in the grains of the tested wheat with genotype AA was higher than that of the tested wheat with genotype GG.

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