SNP molecular marker related to zinc accumulation trait of wheat and breeding application thereof

Abstract

The application discloses a SNP molecular marker related to a wheat zinc accumulation character and breeding application thereof, and belongs to the technical field of gene biology technology.The technical problem to be solved by the application is how to quickly and accurately identify a wheat zinc content character, and therefore provides a SNP site TaZn1-1, TaZn1-2 and a detection material thereof; the average value of the zinc content of a to-be-detected wheat grain of a homozygous type of the genotype A of the TaZn1-1 and a homozygous type of the genotype C of the TaZn1-2 is significantly higher than that of a to-be-detected wheat of a homozygous type of the genotype T of the TaZn1-1 and a homozygous type of the genotype G of the TaZn1-2.The application provides a gene level discrimination basis for the quick screening of high-zinc high-quality wheat germplasm, and has important theoretical value and application prospect for guaranteeing the food safety of China.

Description

Technical Field

[0001] This invention belongs to the field of gene biotechnology, specifically relating to SNP molecular markers related to zinc accumulation traits in wheat and their breeding applications. Background Technology

[0002] Zinc is one of the essential micronutrients for the human body. Zinc deficiency can lead to slow growth, decreased immune function, skin damage, eye diseases, and other illnesses. This is mainly due to the low zinc content in cereal products in people's daily diet.

[0003] Wheat is one of my country's important staple cereal crops. Studies have reported that the average zinc content of wheat grains in major wheat-growing areas of my country is only 30.3 mg / kg, lower than the recommended zinc content of 40–60 mg / kg for human health. However, research on the physiological and molecular mechanisms of zinc accumulation in wheat is still in its early stages. Furthermore, due to the complexity of the wheat genome and the diversity of wheat varieties, research on zinc accumulation in wheat is quite challenging, and systematic studies on wheat varieties with high zinc accumulation have not yet been conducted. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to quickly and accurately identify the zinc content trait in wheat using molecular markers.

[0005] To address the aforementioned issues, this invention provides an application for detecting polymorphisms, genotypes, or haplotypes of SNPs in the wheat genome, wherein the SNPs can be TaZn1-1 and TaZn1-2; TaZn1-1 is an SNP in the wheat genome, corresponding to position 136 of SEQ ID NO: 3, and is either A or T; TaZn1-2 is an SNP in the wheat genome, corresponding to position 159 of SEQ ID NO: 3, and is either C or G; the application can be used for identifying or assisting in the identification of wheat zinc content and / or for wheat breeding.

[0006] In the above applications, the substance may be B1), B2), or B3). B1) The substance described is a primer composition for amplifying wheat genomic DNA fragments including the SNP described above. B2) The substance is a PCR reagent containing the primer composition described in B1). B3) The substance is a kit containing the primer composition described in B1) or the PCR reagent described in B2).

[0007] The present invention also provides a method for identifying or assisting in the identification of zinc content traits in wheat. The method may include detecting the genotype of SNPs in the genome of the wheat to be tested, and identifying or assisting in the identification of zinc content traits in wheat based on the genotypes. The SNPs may be TaZn1-1 and TaZn1-2 as described above.

[0008] This invention also provides the application of the methods described above in wheat breeding.

[0009] The present invention also provides a method for wheat breeding, the method comprising detecting TaZn1-1 and TaZn1-2 in the wheat genome as described above, and selecting wheat with homozygous genotype A of TaZn1-1 and homozygous genotype C of TaZn1-2 as parents for breeding.

[0010] In some embodiments of the present invention, the zinc content of wheat with the AAGG genotype is higher than that of wheat with a non-AAGG genotype, wherein the non-AAGG wheat may be wheat with the TTCC genotype. The wheat with genotype AAGG is a homozygous wheat with genotype A in TaZn1-1 and genotype G in TaZn1-2; the wheat with genotype TTCC is a homozygous wheat with genotype T in TaZn1-1 and genotype C in TaZn1-2.

[0011] The “wheat grain zinc content” is compared under comparable conditions. “Comparable conditions” refer to the same or similar environmental conditions and agronomic practices used to make meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices significantly contribute to or explain any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water, humidity, soil, and nutrients (e.g., nitrogen and phosphorus).

[0012] This invention also provides a product for detecting polymorphisms, genotypes, or haplotypes of SNPs in the wheat genome, wherein the SNP may be the SNP described above, and the product contains the substance described above, wherein the product is as follows: D1), D2), or D3). D1) The product is a primer composition for amplifying wheat genomic DNA fragments including the SNP sites. D2) The product is a PCR reagent containing the primer composition described in D1). D3) The product is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0013] In the above applications and methods, the substance used to detect SNP polymorphisms, genotypes, or haplotypes can be a nucleotide type at the SNP site in the wheat genome determined by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP microarrays. The SNP microarrays include microarrays based on nucleic acid hybridization reactions, microarrays based on single-base extension reactions, microarrays based on allele-specific primer extension reactions, microarrays based on one-step reactions, microarrays based on primer ligation reactions, microarrays based on restriction endonuclease reactions, microarrays based on protein-DNA binding reactions, and microarrays based on fluorescent molecule-DNA binding reactions.

[0014] In the above applications and products, the primer composition used for detection can be a TaZn1 primer pair, which is composed of single-stranded DNA from SEQ ID NO: 1 and SEQ ID NO: 2.

[0015] In the above applications, the primer composition may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Markers include, but are not limited to, dyes; radioactive markers, such as 32P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The marker can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative) or, optionally, charge-neutral. The marker can include a nucleic acid sequence or a protein sequence or a combination thereof, provided that the sequence containing the marker is detectable. In some embodiments, nucleic acids are detected directly without a marker (e.g., direct sequence reading).

[0016] The present invention also provides a PCR reagent containing the primer composition described above for detecting polymorphisms or genotypes of SNP sites in the wheat genome.

[0017] The present invention also provides a kit containing the PCR reagents described above for detecting polymorphisms or genotypes of SNP sites in the wheat genome.

[0018] The present invention also provides a nucleic acid molecule, wherein the nucleotide sequence of the nucleic acid molecule may be SEQ ID NO: 3.

[0019] The indicators for wheat breeding mentioned above include the zinc content of wheat.

[0020] The zinc content of wheat mentioned above specifically refers to the zinc content of wheat grains at maturity.

[0021] Furthermore, the purpose of the wheat breeding includes developing wheat with high zinc content (wheat grain zinc content is higher than that of the parents).

[0022] In the above applications and methods, the wheat can be a homozygous wheat inbred line.

[0023] In the applications and methods described above, wheat inbred lines can be selected as parents for breeding.

[0024] This study investigated the SNP effect of the wheat zinc transporter gene TaZn1 and discovered a SNP that can be used to screen for high-zinc varieties. This provides a genetic basis for the rapid screening of high-zinc, high-quality wheat germplasm and has important theoretical value and application prospects for ensuring my country's food security. Attached Figure Description

[0025] Figure 1 This is a frequency distribution map of zinc content in resource germplasm.

[0026] Figure 2 This refers to the zinc content in the grains corresponding to the two SNPs of TaZn1. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] The 145 wheat samples in the following examples and the 107 wheat samples in Table 2 are disclosed in Supplemental Table 1 of the following literature: Chenyang Hao#, Chengzhi Jiao#, Jian Hou, Tian Li, Hongxia Liu, Yuquan Wang, Jun Zheng, Hong Liu, Zhihong Bi, Fengfeng Xu, Jing Zhao, Lin Ma, Yamei Wang, Uzma Majeed, Xu Liu, Rudi Appels, Marco Maccaferri, Roberto Tuberosa, Hongfeng Lu*, Xueyong Zhang*. Resequencing of 145 landmark cultivars reveals asymmetric sub-genome selection and strong founder genotype effects on wheat breeding in China. Molecular Plant, 2020, 13: 1733-1751. These materials are publicly available from the Institute of Botany, Chinese Academy of Sciences. This biological material is intended solely for the replication of experiments of this invention and should not be used for any other purpose.

[0030] Example 1: Phenotypic Identification of Zinc Content in Wheat Population 1. Cultivation and content determination of wheat germplasm resources 1.1 Determination of Soil Zinc Content Weigh 0.1000 g of sieved soil into a polytetrafluoroethylene digestion tube, and add 1 mL of hydrofluoric acid, 1 mL of aqua regia (concentrated hydrochloric acid: concentrated nitric acid = 3:1), and 1 mL of perchloric acid in sequence. Cap and digest at 150℃ for 5 h. Open the cap halfway through digestion to allow the acidic fumes to completely evaporate. After cooling to room temperature, add 2 mL of nitric acid and continue heating for 3 h until the soil sample is completely clear. Add 2 mL of nitric acid and 10 mL of distilled water, cap, and reconstitute for 4 h to dissolve the residue. Filter through a 0.22 μm filter membrane into a 10 mL plastic centrifuge tube. The soil control is the Tibetan Soil Composition Analysis Standard Material (Institute of Geophysical and Geochemical Exploration, GBW08302).

[0031] 1.2 Cultivation of Wheat Germplasm Resources 145 wheat microcore germplasm resources were planted in Weihui City, Xinxiang City, Henan Province in the winter of 2023. The planting conditions were as follows: watering once each during the overwintering period, jointing period, and grain-filling period; applying basal fertilizer during sowing and land preparation: compound fertilizer (N-P2O5-K2O = [15-15-15]) [30-40] kg per mu; applying urea [10-12] kg per mu as top dressing during the jointing period. The wheat was harvested after reaching maturity.

[0032] 1.3 Determination of Zinc Content in Wheat Grains (1) Select 30 wheat grains harvested in the spring of 2024 for each wheat germplasm. After dehulling by hand, put them into a 5 mL centrifuge tube and add 1 steel ball for grinding. Program: 1800 rpm, 3 cycles, 55 s each time, 35 s rest.

[0033] (2) Accurately weigh 0.2000 g of wheat seeds and put them into a digestion tube.

[0034] (3) Add 1 mL of high-purity nitric acid to the digestion tube (operate in a fume hood), place a bent funnel inside, cover it with a plastic bag, and let it digest overnight in a cold environment.

[0035] (4) On the second day, observe the cold digestion. You can add some nitric acid to ensure that the digestion is complete. Set the digestion oven to 200°C and digest for 9 hours until the liquid is clear.

[0036] (5) After removing it and letting it cool, add distilled water to the clear liquid after digestion and make up to 15 mL using a volumetric immersion tube.

[0037] (6) Shake well, filter through a 0.22 μm filter membrane into a 10 mL plastic centrifuge tube, and determine the zinc content using an inductively coupled plasma mass spectrometer (ICP-MS).

[0038] For each batch, three positive controls were set up using wheat flour composition analysis standard material (Institute of Geophysical and Geochemical Exploration, GBW(E)100493), and two blank controls were set up to remove acid interference.

[0039] Of the 145 samples, seeds were successfully harvested from 127 samples and zinc content was detected. The zinc content determination results are as follows: Figure 1 As shown, the zinc concentration in the grains ranged from 20.92 to 53.53 mg / kg. Figure 1 The horizontal axis represents the interval mean. 20 represents the cadmium concentration in the grain of the germplasm that is ≥19 and <21 mg / kg, 22 represents the zinc concentration in the grain of the germplasm that is ≥21 and <23 mg / kg, and so on.

[0040] 2. Identification of SNP sites in wheat zinc transporter genes: Primers were designed upstream and downstream of the nonsynonymous mutation and PCR amplification was performed using KOD FX enzyme. The primer sequences are shown in Table 1, and the PCR procedure is as follows: (1) Preparation of reaction solution: 2×PCR Buffer for KOD FX25 μL dNTPs 10 μL Forward Primer 1.5 μL Reverse Primer 1.5 μL Template DNA≤50ng (plasmid) / ≤200 ng (DNA / cDNA) KOD FX Enzyme 1 μL Add ddH2O to bring the volume to 50 μL. (2) PCR reaction procedure 94℃ for 2 min Perform the following three steps 40 times. 98℃ for 10 seconds 57℃ for 30 seconds 68℃ 30 s After the loop ends, extend for 7 minutes.

[0041] 68℃ for 7 min 4℃∞ The primer pair for TaZn1 was the forward primer 5'-GACTCGGACGACAAAGTCTC-3' (SEQ ID NO: 1) and the reverse primer 5'-AGCAAACTGACCAGTCGAAG-3' (SEQ ID NO: 2). Sanger sequencing was performed on the PCR amplification results. The PCR product is SEQ ID NO: 3 (593 bp), where the TaZn1-1 site corresponds to position 136 of SEQ ID NO: 3, with polymorphism A or T, denoted by "W"; the TaZn1-2 site corresponds to position 159 of SEQ ID NO: 3, with polymorphism C or G, denoted by "S", as detailed below: 5'--3'.

[0042] In 127 wheat germplasm populations, two non-synonymous mutation SNP sites, Chr4A:41055967 and Chr4A:41055990, were found in the wheat zinc transporter gene TaZn1 (Traes4A03G0096800, Chr4A), named TaZn1-1 and TaZn1-2, respectively. The non-synonymous mutation SNP sites were identified by Sanger sequencing using primers, and the sequencing results are shown in Table 1.

[0043] Table 1. Identification results of non-synonymous mutant SNP sites and zinc content of 127 wheat resource population varieties.

[0044] 3. SNP analysis of wheat zinc transporter gene TaZn1 SNP analysis was performed on the TaZn1 gene to study the relationship between natural variation and phenotype. Grain zinc content data for different SNPs were processed using software. P-values ​​were calculated using Tukey's test. A P-value less than 0.05 was defined as statistically significant; a P-value less than 0.01 was defined as highly statistically significant. Results are shown in Table 2. Figure 2 TaZn1 exists in two haplotypes: TaZn1Hap1 (AG) and TaZn1Hap2 (TC).

[0045] Analysis of the relationship between different SNP haplotypes of the two genes and zinc content revealed that: TaZn1Hap1, containing 90 germplasms, had a grain zinc content of 36.330±5.349 mg / kg; TaZn1Hap2, containing 37 germplasms, had a grain zinc content of 40.432±5.860 mg / kg. The results of the significance analysis are as follows: Figure 2 As shown, the zinc accumulation in the grains of TaZn1Hap2 is significantly higher than that of TaZn1Hap1, which can be used to identify high-zinc superior wheat germplasm.

[0046] Table 2. TaZn1 haplotypes and zinc content in grains

[0047] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. An application for detecting polymorphisms, genotypes, or haplotypes of SNPs in the wheat genome, characterized in that, The SNPs are TaZn1-1 and TaZn1-2; TaZn1-1 is an SNP in the wheat genome, corresponding to position 136 of SEQ ID NO: 3, and is A or T; TaZn1-2 is an SNP in the wheat genome, corresponding to position 159 of SEQ ID NO: 3, and is C or G; The application is for identifying or assisting in the identification of zinc content in wheat and / or for wheat breeding.

2. The application of the substance for detecting SNPs in the wheat genome according to claim 1, characterized in that... The substance is B1), B2), or B3). B1) The substance described is a primer composition for amplifying wheat genomic DNA fragments including the SNP described above. B2) The substance is a PCR reagent containing the primer composition described in B1). B3) The substance is a kit containing the primer composition described in B1) or the PCR reagent described in B2).

3. A method for identifying or assisting in the identification of zinc content in wheat, characterized in that, The method includes detecting the genotype of the SNP described in claim 1 in the genome of the wheat to be tested, and identifying or assisting in the identification of the zinc content of wheat based on the genotype, wherein the SNP is TaZn1-1 or TaZn1-2 as described in claim 1.

4. The application of the method of claim 3 in wheat breeding.

5. A method for wheat breeding, characterized in that, The method includes detecting TaZn1-1 and TaZn1-2 in the wheat genome as described in claim 1, and selecting homozygous wheat with genotype A of TaZn1-1 and homozygous wheat with genotype C of TaZn1-2 as parents for breeding.

6. A product for detecting SNP polymorphisms, genotypes, or haplotypes in the wheat genome, characterized in that, The SNP is the SNP of claim 1, the product contains the substance of claim 1, and the product is as follows: D1), D2), or D3). D1) The product is a primer composition for amplifying wheat genomic DNA fragments including the SNP sites. D2) The product is a PCR reagent containing the primer composition described in D1). D3) The product is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

7. The application according to claim 2 and the product according to claim 6, characterized in that, The primer composition is used to detect that the primer pair is a TaZn1 primer pair; the TaZn1 primer pair consists of single-stranded DNA from SEQ ID NO: 1 and SEQ ID NO:

2.

8. A PCR reagent containing the primer composition of claim 7 for detecting polymorphisms or genotypes of SNP sites in the wheat genome.

9. A kit containing the PCR reagent of claim 8 for detecting polymorphisms or genotypes of SNP sites in the wheat genome.

10. A nucleic acid molecule, characterized in that, The nucleotide sequence of the nucleic acid molecule is SEQ ID NO: 3.

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