Molecular marker for detecting zinc content of wheat grains and application of molecular marker

Abstract

The invention belongs to the technical field of molecular marker detection, and particularly relates to a molecular marker for detecting the zinc content of wheat grains and application of the molecular marker. The method comprises the following steps: detecting g.723 Ggt in a gene TaNAS-6B for regulating and controlling the zinc content of wheat grains; and the site A, such as the 723rd nucleotide of SEQ ID NO: 1, has the nucleotide type of G or A. The invention further provides a method for identifying or assisting in identifying the zinc content character of the wheat grains. The method comprises the following steps: detecting to-be-detected wheat g.723 Ggt; the genotype of the site A is g.723 Ggt; the to-be-detected wheat with the genotype of AA at the site A is high-zinc wheat, and the grain zinc content of the to-be-detected wheat is obviously higher than that of the to-be-detected wheat with the genotype of GG. According to the invention, a dCAPS labeled primer pair is developed and is used for efficiently screening the zinc content of wheat grains. The molecular marker provided by the invention can be used for detecting the genotype of TaNAS-6B, is applied to wheat grain zinc content improvement breeding, and can improve the selective breeding efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker detection, and particularly relates to a molecular marker for detecting zinc content in wheat grains and an application thereof. Background Art

[0002] Zinc is one of the 15 essential trace elements for the human body. Zinc deficiency in the human body can lead to developmental retardation, skin lesions, neurological diseases, and various immune-related diseases. The human body cannot synthesize zinc and needs to obtain it from the outside world through diet. Cereal foods are the main way for humans to take in trace elements. Wheat is one of the world's major food crops. If zinc-rich wheat varieties can be cultivated through biofortification breeding and the zinc content in wheat grains can be increased, zinc deficiency will be eliminated to a large extent, thereby protecting people's lives and health.

[0003] The zinc content in wheat grains is greatly affected by environmental effects, and a large amount of seed samples are required for determination. Therefore, it is impossible to conduct effective determination and screening in the early generations of breeding, resulting in low accuracy and efficiency when using traditional breeding methods to select zinc content. 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 be used to effectively identify genetic loci related to zinc content in wheat grains at the genetic level, and the use of molecular marker-assisted selection strategies for genetic improvement of zinc content in wheat grains can greatly improve the efficiency of breeding selection, thereby shortening the breeding period. Summary of the invention

[0004] The invention provides a molecular marker for detecting the zinc content of wheat grains. The marker can be used for identifying or screening the zinc content of wheat grains, thereby 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, which is the 723rd nucleotide of SEQ ID NO: 1, and the nucleotide type thereof 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 further comprises a restriction endonuclease Hinf I or a sequencing reagent.

[0010] The second aspect of the present invention provides the use of the above product in any of the following:

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

[0012] (2) Identify or assist in identifying the zinc content of wheat grains;

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

[0014] (4) preparing a product for identifying or assisting in identifying or comparing the zinc content of wheat grains to be tested;

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

[0016] The third aspect of the present invention provides a method for identifying the g.723G>A site allele type within the TaNAS-6B gene, which specifically includes the following operating steps: using the genomic DNA of the wheat variety / line to be tested as a template, and using the primer combination in the above product to perform PCR amplification, wherein the length of the amplified product is 1488bp; then using the restriction endonuclease HinfI to digest the above PCR amplification product, and then performing agarose gel electrophoresis to determine the band type; after digestion, the amplified product can be distinguished into two band types of 947bp and 541bp with a GG allele type; after digestion, the amplified product has only one band type of 1488bp with an AA allele type; or sequencing the PCR amplification product to obtain the g.723G>A site allele type within the TaNAS-6B gene.

[0017] The present invention can identify or assist in identifying the zinc content trait of the wheat grain to be tested according to the genotype of the wheat to be tested. Therefore, the fourth aspect of the present invention provides a method for identifying the zinc content trait of wheat grains, comprising: obtaining the genotype of the wheat to be tested by using the above-mentioned method for identifying the g.723G>A site allele type within the TaNAS-6B gene, and then identifying or assisting in identifying the zinc content trait of the wheat grain to be tested according to the genotype, wherein the zinc content of the grain of the wheat to be tested whose g.723G>A site genotype is AA is higher than that of the wheat to be tested whose g.723G>A site genotype is GG; the genotype of GG indicates that the nucleotide type of the g.723G>A site in the wheat genome is a homozygous type of G; the genotype of AA indicates that the nucleotide type of the g.723G>A site in the wheat genome is a homozygous type of A.

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

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

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

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

[0022] 1. The present invention analyzes the cloned wheat grain zinc content regulating gene TaNAS-6B (SEQ ID NO: 1) and finds that the g.723G>A site inside it is significantly associated with the wheat grain zinc content trait. On this basis, the present invention develops and converts the g.723G>A site into a dCAPS marker. Studies have shown that the dCAPS marker of the present invention can be used to detect the genotype of TaNAS-6B, and the method is simple and accurate.

[0023] 2. The molecular marker developed in the present invention can be applied to wheat molecular marker-assisted selection breeding to achieve early screening of wheat grain zinc content traits, which can greatly improve the efficiency of selection breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[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 grain zinc content 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 result of enzyme digestion of the amplified product of the SNP723-F and SNP723-R primer pairs in Example 2 of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; the reagents, carriers and other test materials involved are all commercially available conventional products unless otherwise specified; the test methods, detection methods, etc. involved are all conventional methods unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged. Primer synthesis and sequencing were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0029] Wheat materials involved in the embodiments:

[0030] A natural population consisting of 442 wheat varieties / lines was used for haplotype analysis of the wheat grain zinc content regulatory gene TaNAS-6B and association analysis between the allele type of the g.723G>A site within the TaNAS-6B gene and the grain zinc content phenotype.

[0031] Example 1 Discovery of SNPs Tightly Linked to Wheat Grain Zinc Content Trait within Wheat Grain Zinc Content Regulatory Gene TaNAS-6B

[0032] 1. Group planting

[0033] 442 wheat varieties / lines used for haplotype analysis of TaNAS-6B gene and phenotypic determination of zinc content in grains were planted in Yuanyang Research Base of Henan Agricultural University during the growing season of 2023-2024. A completely randomized block design was adopted. Each planting plot was 2m long and 1.5m wide. There were 12 rows in the plot. Each variety was planted in two rows, with single grain sowing. The spacing between plants in the rows and rows was 20cm and 10cm respectively. The experimental fields were managed according to the standards. There was no drought, lodging, pests and diseases during the entire growth period. After maturity and harvest, the seeds were uniformly threshed, dried and stored.

[0034] 2. Determination of zinc content in seeds

[0035] Flame atomic absorption spectrophotometry was used for determination. Wheat grains were placed in a kraft paper bag, dried at 65°C to constant weight, crushed with a grinder, and stored in a plastic ziplock bag. 1.0 g of the crushed wheat grain sample was accurately weighed in a porcelain crucible using a balance, moved into a muffle furnace after carbonization, and ashed at 550°C for 6 hours to make the ash close to white. Take it out and cool it, put it to room temperature, add 5 mL of nitric acid solution with a volume ratio of 1:1, let it stand, and filter it into a 50 mL volumetric flask with filter paper after the ash is dissolved, and add ddH2O to make it 50 mL. Hitachi ZA-3000 flame atomic absorption spectrophotometer was used for measurement, and a blank control was set at the same time, and three technical repetitions were performed. The zinc content in the grains of the test materials was calculated according to the standard curve. The average of the three measured values ​​was taken as the final investigation value. The measurement results are shown in Table 1.

[0036] Preparation of zinc standard curve: Add zinc standard solution to 7 50mL volumetric flasks, add ddH2O to make the volume 50mL, and make the final concentrations 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 values ​​of zinc content in grains of 442 wheat varieties / lines and genotypes of the g.723G>A locus

[0038]

[0039]

[0040]

[0041]

[0042]

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

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

[0045] The specific steps are as follows:

[0046] ① DNA extraction: DNA of 442 wheat varieties / lines at the seedling stage was extracted 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 2 ml centrifuge tube containing steel balls, freeze them quickly in liquid nitrogen, grind them with a grinder, add 800 μL SLS, shake for 20 min, and mix them thoroughly; the SLS contains 288 mM NaCl, 200 mM Tris-HCl, 25 mM EDTA, and 0.5% SDS;

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

[0050] (3) Oscillate for 10 min 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 for precipitation;

[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, dry under vacuum, and add 100 μL TE to dissolve the precipitate to obtain wheat leaf genomic DNA.

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

[0055] Design PCR primers. TaNAS-6B-F and TaNAS-6B-R were designed at the positions of the start codon ATG and the stop codon TAG, respectively. The specific information is as follows:

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

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

[0058] PCR amplification was performed using the DNA of 442 wheat varieties / lines as templates using the primer pair 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 TaNAS-6B gene by primer pair 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. Primer pair composed of TaNAS-6B-F and TaNAS-6B-R for PCR amplification of TaNAS-6B gene

[0062]

[0063] The PCR products were subjected to 1% agarose gel electrophoresis, and after the target band was detected and amplified, they were sent to Shanghai Bioengineering for sequencing.

[0064] The sequencing results were analyzed and the results were as follows Figure 1As shown, among the 442 wheat varieties / lines tested, there are 9 SNP sites in the coding region of the TaNAS-6B gene, which are located at 92bp, 384bp, 390bp, 442bp, 559bp, 648bp, 684bp, 723bp and 732bp from the first start codon ATG in SEQ ID NO:1, and the TaNAS-6B gene can be divided into four haplotypes based on these 9 SNP sites, named Hap1, Hap2, Hap3 and Hap4, respectively.

[0065] The four haplotypes of TaNAS-6B were combined with the phenotypic values ​​of grain zinc content to analyze the results. Figure 2 As shown, the grain zinc content of wheat varieties / lines with Hap2 type is significantly higher than that of the other three haplotypes. Further analysis found that the variable site g.723G>A located at the 723bp from the first start codon ATG in SEQ ID NO:1 is a specific site that distinguishes Hap3 from the other three haplotypes, and this site is closely linked to the grain zinc content.

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

[0067] The sequence of the g.723G>A site in the TaNAS-6B gene was analyzed, and it was found that the site was located on the recognition sequence of the restriction endonuclease Hinf I. When the site was GG genotype, it could be recognized and cut by Hinf I, but when the site was AA genotype, it could not be recognized by Hinf I. Based on this, the dCAPS marker was developed. First, based on the Chinese Spring reference genome sequence, a specific primer pair SNP723-F and SNP723-R were designed to amplify a DNA fragment containing the g.723G>A site. The length of the amplified fragment was 1488bp, and the sequence of the amplified product was shown in SEQ ID NO:2. The nucleotide sequence of the primer pair is as follows:

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

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

[0070] DNA of some lines were randomly selected from 442 wheat varieties / lines and amplified using the above primer pairs. 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 where g.723G>A is located

[0072]

[0073]

[0074] Table 5. PCR amplification procedure of the primer pair composed of SNP723-F and SNP723-R for the DNA fragment where g.723G>A is located

[0075]

[0076] 20 μL of the amplified product was taken out and digested with restriction endonuclease HinfI at 37° C. The digestion reaction system is shown in Table 6.

[0077] Table 6. Reaction system of amplification products by primer pair SNP723-F and SNP723-R

[0078] Components volume PCR products 20μL 10×buffer 3μL Hinf I 1μL <![CDATA[ddH2O]]> To 30μL

[0079] The above enzyme digestion products were subjected to 1% agarose gel electrophoresis to detect whether the DNA fragments amplified by the SNP723-F and SNP723-R primer pairs could be digested by enzymes. Figure 3 As shown in the figure, the DNA fragment of the wheat line with the GG genotype at the g.723G>A site can be successfully cut into two fragments of 947 bp and 541 bp in length, while the DNA fragment of the wheat line with the AA genotype at the g.723G>A site cannot be cut. This result shows that the dCAPS marker can be used to distinguish the allele type of the g.723G>A site.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A test TaNAS-6B The product of the g.723 G>A site within the gene is characterized by: The products include: (1) For detection TaNAS-6B Primer composition for the genotype of the g.723 G>A site within the gene, wherein the g.723 G>A site is a TaNAS-6B A SNP site within the gene is the 723rd nucleotide of SEQ ID NO: 1, and the nucleotide type is G or A; the primer composition comprises: SNP723-F :5’- CTCAAAGCCGGACGTCAGAG-3’; SNP723-R :5’- TCGTGGACCAACAGAGAACC-3’; (2) A reagent or kit comprising the primer composition described in (1).

2. Use of the product according to claim 1 in any of the following items, characterized in that: (1) Identification of wheat TaNAS-6B Allele type of g.723 G>A site within the gene; (2) Identify or assist in identifying the zinc content of wheat grains; (3) Screening or assisting in the screening of wheat plants, lines, strains or varieties with high zinc content in wheat grains; (4) Preparing products for identifying or assisting in identifying or comparing the zinc content of wheat grains to be tested; (5) Molecular marker-assisted selection breeding of wheat.

3. Identification TaNAS-6B The method for determining the allele type of the g.723 G>A site within a gene is characterized in that The method comprises the following steps: using the genomic DNA of the wheat variety / line to be tested as a template, and performing PCR amplification using the primer combination in the product of claim 1; Then, restriction endonucleases were used Hinf I. The PCR amplification product is digested by enzyme, and then subjected to agarose gel electrophoresis to determine the band type. The amplification product can be distinguished into two band types of 947 bp and 541 bp after enzyme digestion, which has the GG allele type; the amplification product has only one band type of 1488 bp after enzyme digestion, which has the AA allele type; or the PCR amplification product is sequenced to obtain TaNAS-6B Allele type of g.723 G>A site within the gene.

4. A method for identifying the zinc content of wheat grains, characterized in that: include: The genotype of the wheat to be tested is obtained by the method described in claim 3, and then the zinc content trait of the wheat grain to be tested is identified or auxiliary identified based on the genotype; the zinc content of the grain of the wheat to be tested whose genotype at the g.723 G>A site is AA is higher than that of the wheat to be tested whose genotype at the g.723 G>A site is GG.

5. A method for breeding new high-quality wheat varieties / lines, characterized in that: The steps include: (1) Using the method of claim 3 to detect the TaNAS-6B The allele type of the g.723 G>A site within the gene; (2) Select wheat breeding materials with the AA allele as the paternal / maternal parent for subsequent breeding.

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