Wheat grain zinc and iron content marking method and application

Abstract

The invention belongs to the technical field of wheat germplasm identification, particularly relates to a wheat grain zinc and iron content marking method and application, discloses four SNP (Single Nucleotide Polymorphism) sites QGZn-4B, QGZn-7B, QGFe-2A and QGFe-3B which are stably associated with the zinc content and the iron content of wheat grains, and solves the core pain points of poor molecular marker stability and indefinite association in the prior art. And a reliable genetic target is provided for accurate identification of the zinc and iron content of wheat. According to the invention, a KASP specific primer combination developed for four SNP loci is also adopted, so that efficient genetic typing of a wheat material is realized, a complicated sequencing process is not needed, the operation is simple and convenient, the specificity is high, and the problems of long detection period, complicated operation and high cost of a traditional molecular marker are effectively avoided. The blank of efficient tools in current wheat zinc and iron content molecular marker assisted breeding is effectively filled, and the method has important significance in promoting innovation and upgrading of a wheat nutritional quality breeding technology.

Description

Technical Field

[0001] This invention belongs to the field of wheat germplasm identification technology, specifically relating to a method for marking the zinc and iron content of wheat grains and its application. Background Technology

[0002] Zinc and iron are both essential trace elements for the human body, participating in various physiological metabolic processes and playing a vital role in maintaining human health. Wheat, as one of the world's major food crops, is an important dietary source of zinc and iron for humans, and the zinc and iron content in its grains directly affects the level of dietary nutrition. Therefore, cultivating wheat varieties with high zinc and iron content is an effective way to improve micronutrient deficiencies and enhance the nutritional quality of grains.

[0003] Currently, zinc and iron content in wheat grains are typical quantitative traits, controlled by multiple genes and easily affected by environmental factors. Traditional breeding methods rely on phenotypic identification to screen for high-content materials, which suffers from problems such as long cycles, low efficiency, and insufficient accuracy, making it difficult to meet the demand for rapid breeding of high-quality wheat varieties. With the development of molecular marker technology, genome-wide association analysis has become an important means of discovering gene loci related to quantitative traits in crops. This technology can accurately locate molecular markers related to zinc and iron content in wheat grains, providing a theoretical basis for marker-assisted breeding.

[0004] However, no KASP functional markers have yet been developed targeting stable association sites for zinc and iron content in wheat grains, thus failing to provide an efficient tool for the rapid breeding of high-zinc and high-iron wheat varieties. Therefore, it is urgent to identify stable association SNP sites related to zinc and iron content in wheat grains, develop corresponding KASP markers, and establish an accurate and efficient method for marking and detecting zinc and iron content in wheat grains. This would solve the problems of low phenotypic identification efficiency and insufficient practicality of molecular markers in traditional breeding, and accelerate the breeding process of high-nutritional-quality wheat varieties. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for labeling zinc and iron content in wheat grains. This method can accurately identify four SNP sites that are stably associated with zinc and iron content in wheat grains. Furthermore, by developing specific KASP markers, it enables efficient and high-throughput genotyping of the target sites, providing a reliable molecular detection tool for the targeted breeding of high-nutritional-quality wheat varieties.

[0006] The specific technical solution adopted in this invention is as follows: A method for labeling zinc and iron content in wheat grains, wherein the labeling method is used to identify SNP sites associated with zinc and iron content in wheat grains. The SNP locus is QGZn-4B, located on wheat chromosome 4B, with a physical location of 663193293. The average zinc content is higher for genotype A than for genotype C. Alternatively, the locus is QGZn-7B, located on wheat chromosome 7B, with a physical location of 581709389. When the genotype is C, the average zinc content is higher than that of the genotype T. Alternatively, the locus is QGFe-2A, located on wheat chromosome 2A, with a physical location of 708057740, and the average iron content is higher in genotype T than in genotype C. Alternatively, the locus is QGFe-3B, located on wheat chromosome 3B at physical location 447303598, and the mean iron content is higher in genotype C than in genotype T.

[0007] The method further includes a KASP primer combination for identifying the SNP site, wherein, KASP primer combinations corresponding to QGZn-4B: F1: GAAGGTGACCAAGTTCATGCTCCAACCAGAAAAGGTCTGATCTTTACT; F2: GAAGGTCGGAGTCAACGGATTCAACCAGAAAAGGTCTGATCTTTACG; R: AGCTCAAACAACAATTGCAAATCAT; KASP primer combinations corresponding to QGZn-7B: F1: GAAGGTGACCAAGTTCATGCTGTGCTTGCTTCTGCTGCTCTT; F2: GAAGGTCGGAGTCAACGGATTGTGCTTGCTTCTGCTGCTCTC; R: GTAGTGACAAGAACAATAGCAGTGTGAT; KASP primer combinations corresponding to QGFe-2A: F1: GAAGGTGACCAAGTTCATGCTGGTCTTCTGCTTGAAGCGGC; F2: GAAGGTCGGAGTCAACGGATTAAGGTCTTCTGCTTGAAGCGGT; R: GGACGACCAACCGGTAGATCA; KASP primer combinations corresponding to QGFe-3B: F1: GAAGGTGACCAAGTTCATGCTGGTGGTCAATGATTGGTCAACTA; F2:GAAGGTCGGAGTCAACGGATTGGTGGTCAATGATTGGTCAACTG; R:GCCGGCACACTGTAGATCACA.

[0008] An application of a method for labeling zinc and iron content in wheat grains, wherein the application is in a reagent or kit containing a combination of KASP primers for the SNP site.

[0009] The beneficial effects of this invention are: This invention discloses four SNP sites, QGZn-4B, QGZn-7B, QGFe-2A, and QGFe-3B, that are stably associated with the zinc and iron content of wheat grains. This solves the core problems of poor molecular marker stability and unclear association in the prior art, and provides reliable genetic targets for the accurate identification of zinc and iron content in wheat.

[0010] This invention also developed a KASP-specific primer combination targeting four SNP loci, enabling efficient genotyping of wheat materials without the need for complex sequencing procedures. The method is simple to operate, highly specific, and effectively avoids the problems of long detection cycles, cumbersome operations, and high costs associated with traditional molecular marker detection. This effectively fills the gap in efficient tools for molecular marker-assisted breeding of wheat zinc and iron content, and is of great significance for promoting the innovation and upgrading of wheat nutritional quality breeding technology. Attached Figure Description

[0011] Figure 1 Manhattan and QQ graphs for wheat GZnC; Figure 2 Manhattan and QQ graphs of wheat GFeC; Figure 3 ABCD represent the KASP classification diagrams of QGZn-4B (SNP1), QGZn-7B (SNP2), QGFe-2A (SNP3), and QGFe-3B (SNP4), respectively. Detailed Implementation

[0012] The present invention will be further described below with reference to the accompanying drawings and specific embodiments: Specific Implementation Example 1: Selection of SNP sites.

[0013] This invention used 185 natural population data for genome-wide association analysis. These data mainly came from wheat varieties in the Huang-Huai winter wheat region, the northern winter wheat region, the middle and lower reaches of the Yangtze River winter wheat region, and the southwestern winter wheat region of my country.

[0014] Wheat varieties were planted in October 2021 and October 2022 at the Dishan Experimental Station (22SZR, 23SZR) of the Institute of Grain and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences, and the Gaoyi Experimental Station (23YZR) of the Chinese Academy of Agricultural Sciences. Each material was planted in one row, 1 m long, with a row spacing of 25 cm. Two replicates were set up for each planting site, and the average of the two replicates was used. Harvesting was carried out at maturity when the grains were completely dry, and threshing was done manually to avoid potential mineral contamination. Approximately 15 g of the sample was weighed, and the Zn concentration (GZnC) and Fe concentration (GFeC) of the wheat grains were determined using an X-ray fluorescence spectrometer (EDXRF, model X-Supreme 8000, Oxford Instruments). The best linear unbiased prediction (BLUP) value was obtained considering environmental factors, and a total of four environments were included, as shown in Table 1.

[0015] Table 1. Content data measured from 185 natural population materials.

[0016] Based on the genotyping results, the genotyping data from the 660K SNP chip were quality controlled. PLINK software was used to remove SNP markers with a minimum allele frequency (MAF) < 0.05 and missing data > 15%. Genome-wide association analysis (GWAS) was performed using a mixed linear model (MLM) in TASSEL 5.0 software, with a threshold set at p < 0.001 for single nucleotide polymorphisms (SNPs) that were detected as stably associated in two or more environments. A total of 177,632 SNPs were available for subsequent GWAS analysis. GWAS was performed by combining the phenotypic data of zinc and iron content in wheat grains under four environmental conditions with the genotyping results from the 660K chip. Figure 1 and Figure 2 Four stable associated SNP sites were identified (Tables 2 and 3). The physical locations are based on the sequences in Chinese Spring genome v1.0.

[0017] Table 2. Information on SNP sites that are significantly associated with Zn / Fe content in wheat grains.

[0018] Table 3. Statistical analysis of the relationship between allele variation type and wheat grain content.

[0019] Note: Statistical analysis was performed using the t-test (P < 0.05 indicates a significant difference; P < 0.01 indicates a highly significant difference).

[0020] Specific embodiment 2: Development and verification of KASP markers for SNP sites related to zinc and iron content in wheat grains.

[0021] 168 natural population materials were used for locus validation of zinc and iron content in grains.

[0022] (1) Take an appropriate amount of young wheat leaves into a 2 mL centrifuge tube, freeze them quickly with liquid nitrogen, and grind the leaves into powder on a grinder. (2) Add 800 μL of TPS to the broken leaf tissue, incubate in a water bath at 80°C for 30 min, shake several times every 10 min, and centrifuge at 12000 r / min for 10 min at room temperature. (3) Immediately after centrifugation, take 500 μL of the supernatant, add an equal volume of pre-cooled isopropanol, shake gently up and down a few times, and white flocculent matter will be produced. Centrifuge at 12000 r / min for 10 min. (4) Discard the supernatant, and wash the precipitate with 300 μL of 75% ethanol until the precipitate is transparent; (5) Let it stand in a fume hood to air dry. After the ethanol has evaporated, add 100 μL ddH2O to dissolve the DNA and store it at 20℃ for later use.

[0023] DNA quality testing and dilution DNA was detected using 1% agarose gel electrophoresis to ensure the integrity of the wheat genomic DNA. DNA concentration was measured using a NanoDrop 2000, and the DNA was diluted to a working concentration of approximately 50 ng / μL and stored at 20°C for later use.

[0024] KASP markup development KASP primers were designed based on SNP sites. Forward primer F1 was fitted with a linker for the FAM sequence “5'-GAAGGTGACCAAGTTCATGCT-3'”, and forward primer F2 was fitted with a linker for the HEX sequence “5'-GAAGGTCGGAGTCAACGGATT-3'”. The primer sequences are shown in Table 4.

[0025] Table 4 KASP primer sequences

[0026] KASP Reaction System and Procedure Dilute the primers to 100 mM with ddH2O and prepare the following solution: 12 μL of forward primer F1, 12 μL of forward primer F2, 30 μL of reverse primer R, and 46 μL of ddH2O, for a total of 100 μL. This solution will be used as the working solution for KASP-labeled primers. Store at 20°C for later use.

[0027] The reaction system consists of 2.5 μL of HiGeno 2x Probe Mix (Beijing Jiacheng Biotechnology Co., Ltd.), 0.07 μL of primer working solution, 1 μL of template DNA, and 1.5 μL of ddH2O, for a total of 5 μL.

[0028] Follow the KASP amplification procedure below (1) Pre-denaturation at 95℃ for 10 min; (2) Denaturation at 95℃ for 20 s; (3) Anneal at 61℃ for 40 s (decreasing by 0.6℃ per cycle), repeat steps 2 and 3 10 times; (4) Denaturation at 95℃ for 20 s; (5) Anneal at 55℃ for 40 s, repeat steps 4 and 5 35 times; (6) Store at 25℃ and detect the signal.

[0029] After PCR amplification, the information was read on a quantitative PCR instrument (BIO-RAD). Fluorescent signals with FAM tags (orange) clustered near the X-axis, fluorescent signals with HEX tags (blue) clustered near the Y-axis, and the rest (green) were heterozygous. Figure 3 ).

[0030] Based on KASP marker testing, the genotyping of 168 wheat varieties is shown in Table 5 below.

[0031] Table 5 KASP classification results

[0032] Note: NA represents missing data, H represents Heterozygote, and U represents Undetermined.

[0033] Specific Example 3: Application of reagents and kits containing KASP primer combinations in the identification of zinc and iron content in wheat grains.

Claims

1. A method for labeling zinc and iron content in wheat grains, wherein the labeling method is used to identify SNP sites related to zinc and iron content in wheat grains, characterized in that: The SNP locus is QGZn-4B, located on wheat chromosome 4B, with a physical location of 663193293. The average zinc content is higher for genotype A than for genotype C. Alternatively, the locus is QGZn-7B, located on wheat chromosome 7B, with a physical location of 581709389, and the average zinc content is higher in genotype C than in genotype T. Alternatively, the locus is QGFe-2A, located on wheat chromosome 2A, with a physical location of 708057740, and the average iron content is higher in genotype T than in genotype C. Alternatively, the locus is QGFe-3B, located on wheat chromosome 3B at physical location 447303598, and the mean iron content is higher for genotype C than for genotype T.

2. The method for marking zinc and iron content in wheat grains according to claim 1, characterized in that: The method further includes a KASP primer combination for identifying the SNP site, wherein, KASP primer combinations corresponding to QGZn-4B: F1: GAAGGTGACCAAGTTCATGCTCCAACCAGAAAAGGTCTGATCTTTACT; F2: GAAGGTCGGAGTCAACGGATTCAACCAGAAAAGGTCTGATCTTTACG; R: AGCTCAAACAACAATTGCAAATCAT; KASP primer combinations corresponding to QGZn-7B: F1: GAAGGTGACCAAGTTCATGCTGTGCTTGCTTCTGCTGCTCTT; F2: GAAGGTCGGAGTCAACGGATTGTGCTTGCTTCTGCTGCTCTC; R: GTAGTGACAAGAACAATAGCAGTGTGAT; KASP primer combinations corresponding to QGFe-2A: F1: GAAGGTGACCAAGTTCATGCTGGTCTTCTGCTTGAAGCGGC; F2: GAAGGTCGGAGTCAACGGATTAAGGTCTTCTGCTTGAAGCGGT; R: GGACGACCAACCGGTAGATCA; KASP primer combinations corresponding to QGFe-3B: F1: GAAGGTGACCAAGTTCATGCTGGTGGTCAATGATTGGTCAACTA; F2:GAAGGTCGGAGTCAACGGATTGGTGGTCAATGATTGGTCAACTG; R:GCCGGCACACTGTAGATCACA.

3. An application based on the method of claim 2, characterized in that: The application is an application in a reagent or kit that contains a combination of KASP primers for the SNP site.

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