A SNP molecular marker simultaneously linked to QTLs for copper and magnesium content in wheat and its application

Abstract

The present invention discloses a SNP molecular marker simultaneously linked to QTLs for copper and magnesium content in wheat and its application, wherein the polymorphism of the SNP molecular marker is A / G, and the SNP molecular marker is co-localized with QTLs QGrCuc.sau‑4A for copper content in wheat and QGrMgc.sau‑4A for magnesium content in wheat on the short arm of chromosome 4A of the wheat genome, and the SNP molecular marker is obtained by amplifying the primer combination shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The present invention discloses QTLs QGrCuc.sau‑4A for copper content in grains, QTLs QGrMGc.sau‑4A for magnesium content and SNP molecular markers KASP‑4A‑sau1, which can quickly screen plants with the loci, facilitate molecular assisted breeding of wheat, and improve the working efficiency of breeding.

Description

Technical Field

[0001] The invention relates to the technical field of wheat molecular genetic breeding, and in particular to a SNP molecular marker simultaneously linked to QTLs for copper and magnesium contents in wheat and an application thereof. Background Art

[0002] Wheat (Triticum sp.) is a widely grown food crop worldwide and the main food source for 35-40% of the world's population. Faced with the increase in global population, most breeders focus their research goals on high wheat yields, ignoring that the reduction in trace element content caused by increased wheat yields has become a global problem. In poor areas where people have limited diets and industrial investment in fortification is insufficient, biofortification has become the most promising, cost-effective and sustainable strategy to increase the copper and magnesium content of wheat grains to solve malnutrition.

[0003] Copper and magnesium are both essential mineral elements for humans and plants, and are involved in many physiological and biochemical activities. Copper and magnesium deficiency in the human body can lead to skeletal dysplasia. Copper and magnesium are important components of chlorophyll. Almost all copper in leaves is present in chloroplasts, which plays a stabilizing role in chloroplasts to prevent chlorophyll from being destroyed. About 75% of magnesium in plants is involved in protein synthesis in leaves, and 15-20% of total magnesium is related to the synthesis of chlorophyll pigments. Magnesium deficiency in crops can lead to the destruction of chloroplast structure and a decrease in the number of thylakoids. Studies have shown that in the process of improving common wheat, due to the use of a few backbone parent resources, the genetic diversity between varieties has gradually decreased, and the copper and magnesium content of grains is limited by a narrow genetic basis, which has obvious adverse effects on future wheat improvement breeding. However, a large number of untapped genetic resources in wheat relatives can effectively help us face the challenges of future wheat production. Accelerating the use of unique genetic resources in relatives is very important for increasing the copper and magnesium content of common wheat grains. Different genotypes of wheat have different abilities to absorb and utilize nutrients. Therefore, we are committed to the study of nutritional genetic positioning of copper and magnesium efficient genotypes, improving and enhancing the efficiency of wheat in absorbing and utilizing soil copper and magnesium elements, and taking effective measures to cultivate copper and magnesium efficient wheat varieties.

[0004] Single nucleotide polymorphism (SNP) markers refer to DNA sequence polymorphisms caused by the variation of a single nucleotide at the genome level. They have the advantages of large number, wide distribution, uneven distribution in a single gene and the entire genome, and easy estimation of SNP allele frequency. They are the most numerous and most promising molecular markers in current genetic marker research. Among them, 55K SNP chips, 90K SNP chips and 660K SNP chips have been widely used in wheat.

[0005] Kompetitive Allele Specific PCR (KASP) is a new low-cost, high-throughput genotyping technology developed by LGC (Laboratory of the Government Chemist). It accurately performs biallelic typing of SNPs and InDel sites through specific matching of primer terminal bases. It is widely used in molecular marker-assisted selection of crops such as rice, wheat, and soybeans.

[0006] In recent years, there have been reports on QTL positioning of copper and magnesium content in wheat grains. However, there are currently few molecular markers that are tightly linked to the copper and magnesium content traits in grains and can be used for actual molecular breeding. Therefore, it is of great significance to develop a molecular marker that can be tightly linked to the QTLs for both copper and magnesium content in grains, to use this marker to screen wheat lines with suitable copper and magnesium content in grains through molecular biotechnology, and to use this molecular marker for wheat breeding. Summary of the invention

[0007] In order to solve the above technical problems, the purpose of the present invention is to provide a SNP molecular marker simultaneously linked to the QTL for copper and magnesium content in wheat and its application, by which the SNP molecular marker KASP-4A-sau1 is tightly linked to the QTL for copper content in wheat grains QGrCuc.sau-4A and the QTL for magnesium content in wheat grains QGrMgc.sau-4A, the QTL for copper and magnesium content in wheat grains can be accurately tracked, and the copper and magnesium content in wheat grains can be predicted, thereby significantly improving the efficiency of selection and identification of suitable grain copper and magnesium content in wheat, being able to quickly screen out wheat varieties or lines with suitable grain copper and magnesium content for breeding, and accelerating the process of molecular genetic breeding of wheat.

[0008] The technical solution of the present invention to solve the above technical problems is as follows:

[0009] A SNP molecular marker simultaneously linked to QTLs for copper and magnesium content in wheat, the SNP molecular marker polymorphism is A / G, and the SNP molecular marker is co-localized with the wheat copper content QTL QGrCuc.sau-4A and the magnesium content QTL QGrMgc.sau-4A on the short arm of chromosome 4A of the wheat genome. The SNP molecular marker is amplified by a primer combination as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0010] The beneficial effects of the present invention are as follows: the present invention discloses a QTL QGrCuc.sau-4A for copper content in wheat grains and a QTL QGrMgc.sau-4A for magnesium content in wheat grains, and the two QTLs are co-located on the short arm of chromosome 4A of tetraploid wheat, and can significantly increase the contents of copper and magnesium in wheat grains, and have a high utilization value in wheat breeding for regulating the copper and magnesium contents in grains; in addition, the present invention also discloses a SNP molecular marker KASP-4A-sau1 that can simultaneously and accurately detect the QTL QGrCuc.sau-4A for copper content in wheat grains and the QTL QGrMgc.sau-4A for magnesium content based on a fluorescent quantitative PCR platform, and the SNP molecular marker is a colinear marker, and has accurate and efficient detection and convenient amplification.

[0011] A primer combination for amplifying a SNP molecular marker, the primer combination comprising two upstream primers as shown in SEQ ID NO.1 and SEQ ID NO.2 and a downstream primer as shown in SEQ ID NO.3.

[0012] The primer combination is used in preparing a chip for identifying wheat genes or gene fragments thereof.

[0013] The primer combination is used in preparing a kit for identifying wheat genes or gene fragments thereof.

[0014] A chip for identifying wheat genes or gene fragments thereof comprises the above primer combination.

[0015] A kit for identifying wheat genes or gene fragments thereof comprises the above primer combination.

[0016] The beneficial effects of the present invention are as follows: the present invention provides a chip and a kit for identifying wheat genes or gene fragments, which are prepared by amplification primers based on the SNP molecular marker KASP-4A-sau1. The chip or the kit can be used to detect the QTLs of grain copper and magnesium content on wheat chromosome 4A by fluorescent quantitative PCR technology, and plants with the locus can be quickly screened, thereby facilitating molecular assisted breeding of wheat.

[0017] The above-mentioned SNP molecular marker or primer combination or chip or kit is used in wheat molecular genetic breeding or cultivation of transgenic wheat or wheat germplasm resource improvement or screening of wheat lines / variety or genetic analysis and positioning of wheat copper and magnesium content genes.

[0018] The beneficial effects of the present invention are as follows: the SNP molecular marker KASP-4A-sau1 used in the present invention can be closely linked to the grain copper content QTL QGrCuc.sau-4A and the magnesium content QTL QGrMgc.sau-4A on wheat 4A, and can be used to locate the copper and magnesium content traits of wheat grains, thereby being applied to molecular genetic breeding of wheat, breeding of transgenic wheat, improvement of wheat germplasm resources, screening of wheat lines / varieties, and genetic analysis and positioning of wheat copper and magnesium content genes, eliminating plants with low grain copper and magnesium contents during the breeding process, improving breeding work efficiency, and providing a basis for research on wheat grain copper and magnesium content genes.

[0019] A method for screening wheat lines containing both copper content QTL QGrCuc.sau-4A and magnesium content QTL QGrMgc.sau-4A, comprising the following steps:

[0020] The genomic DNA of the plant sample to be tested was used as a template, and the above-mentioned primer combination was used for fluorescence quantitative PCR amplification. The amplification results were then used for genotyping, and the plants that could read the fluorescent group labeled with the sequence such as SEQ ID NO.2 were identified as wheat lines containing both the copper content QTL QGrCuc.sau-4A and the magnesium content QTL QGrMgc.sau-4A.

[0021] The beneficial effects of the present invention are as follows: the present invention can rapidly locate the copper and magnesium content traits of wheat grains by using a primer combination for amplifying SNP molecular markers through fluorescent PCR technology, and rapidly screen wheat lines containing both the copper content QTL QGrCuc.sau-4A and the magnesium content QTL QGrMgc.sau-4A, thereby screening out plants with grains having appropriate copper and magnesium contents.

[0022] Furthermore, the reaction system for fluorescence quantitative PCR amplification was: 5 μL Master Mix, 5 ng template DNA, 1.4 μL mixed primers, and ddH2O added to a total volume of 10 μL.

[0023] Furthermore, the mixed primer consists of an upstream primer solution shown in SEQ ID NO.1, an upstream primer solution shown in SEQ ID NO.2, a downstream primer solution shown in SEQ ID NO.3 and ddH2O.

[0024] Furthermore, the volume ratio of the upstream primer solution shown in SEQ ID NO.1, the upstream primer solution shown in SEQ ID NO.2, the downstream primer solution shown in SEQ ID NO.3 and ddH2O is (100-150): (100-150): (250-350): (400-500).

[0025] Preferably, the volume ratio of the upstream primer solution shown in SEQ ID NO.1, the upstream primer solution shown in SEQ ID NO.2, the downstream primer solution shown in SEQ ID NO.3 and ddH2O is 120:120:300:460.

[0026] Furthermore, the concentration of the upstream primer solution shown in SEQ ID NO.1 is 8-12 ng / μL, the concentration of the upstream primer solution shown in SEQ ID NO.2 is 8-12 ng / μL, and the concentration of the downstream primer solution shown in SEQ ID NO.3 is 8-12 ng / μL.

[0027] Preferably, the concentration of the upstream primer solution shown in SEQ ID NO.1 is 10 ng / μL, the concentration of the upstream primer solution shown in SEQ ID NO.2 is 10 ng / μL, and the concentration of the downstream primer solution shown in SEQ ID NO.3 is 10 ng / μL.

[0028] Furthermore, the reaction procedure of fluorescence quantitative PCR amplification was as follows: pre-denaturation at 94 °C for 15 min, followed by denaturation at 94 °C for 20 s and annealing / extension at 61 °C for 60 s, for a total of 10 cycles; and finally denaturation at 94 °C for 20 s and annealing / extension at 55 °C for 60 s, for a total of 26 cycles.

[0029] The present invention has the following beneficial effects:

[0030] (1) The present invention discloses a QTL for copper content in grains QGrCuc.sau-4A and a QTL for magnesium content QGrMGc.sau-4A co-localized on the short arm of wheat chromosome 4A from wild emmer wheat "LM001". The QTL can significantly increase the copper and magnesium contents in wheat grains and has a high utilization value in wheat breeding for regulating the copper and magnesium contents in grains.

[0031] (2) The present invention discloses a SNP molecular marker KASP-4A-sau1 which is tightly linked to the grain copper content QTL QGrCuc.sau-4A and the magnesium content QTL QGrMGc.sau-4A. The marker is a colinear marker with accurate and efficient detection and convenient and stable amplification. The marker characteristics tightly linked to the grain copper content QTL QGrCuc.sau-4A and the magnesium content QTL QGrMGc.sau-4A have high accuracy in molecular marker-assisted selection, can significantly improve the selection and identification efficiency of wheat varieties with higher grain copper and magnesium contents that are adapted to different environments, and has a high success rate. The marker can be used to detect the grain copper and magnesium content QTL on wheat chromosome 4A, quickly screen plants with the loci, and thus facilitate molecular-assisted breeding of wheat.

[0032] (3) The present invention discloses a method for screening wheat lines containing both copper content QTL QGrCuc.sau-4A and magnesium content QTL QGrMgc.sau-4A by using fluorescent quantitative PCR technology and SNP molecular marker KASP-4A-sau1. By locating the copper and magnesium content traits of wheat grains, plants with low copper and magnesium contents in grains are eliminated during the breeding process, thereby improving the efficiency of breeding work and providing a basis for the study of copper and magnesium content genes in wheat grains. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The co-localization of the QTL QGrCuc.sau-4A for copper content in wheat grains and the QTL QGrMgc.sau-4A for magnesium content on chromosome 4A in Example 1;

[0034] Figure 2 The fluorescence reading results of the molecular marker KASP-4A-sau1 detected in the recombinant inbred line of wheat "Ailanmai"×"LM001" in Example 1, wherein HEX (square) fluorescence represents the line with higher grain copper and magnesium content, and FAM (circle) fluorescence represents the line with lower grain copper and magnesium content; the black diamond fluorescence represents the blank control;

[0035] Figure 3 The fluorescence readings of the recombinant inbred lines of wheat "LM001" × "PI 503554" detected by the molecular marker KASP-4A-sau1 in Example 2, wherein HEX (square) fluorescence represents the lines with higher grain copper and magnesium contents, FAM (circle) fluorescence represents the lines with lower grain copper and magnesium contents, triangle fluorescence represents the heterozygous lines, and black diamond fluorescence represents the blank control. DETAILED DESCRIPTION

[0036] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0037] The wheat lines "Ailanmai", "LM001" and "PI 503554" used in the following examples were all provided by the Wheat Institute of Sichuan Agricultural University.

[0038] Example 1: Obtaining QTL QGrCuc.sau-4A for copper content and QTL QGrMgc.sau-4A for magnesium content in wheat grains and their SNP molecular marker KASP-4A-sau1

[0039] (1) The wheat line “Ailanmai” was used as the female parent and the wheat line “LM001” as the male parent for hybridization to obtain the hybrid F1 generation. The F2 generation was then obtained through self-pollination of the F1 generation plants. The F9 generation recombinant inbred line (RIL) population containing 121 lines was obtained by single seed propagation to form the genetic mapping population.

[0040] (2) Phenotypic identification of copper and magnesium content in grains of recombinant inbred line populations

[0041] A field experiment was conducted, with a total of 5 ecological sites (Wenzhou in 2020, Ya'an in 2021, Chongzhou in 2021, Wenzhou in 2022, and Chongzhou in 2022). Each material was planted in one row at each environmental site (row length 1.5 m, row spacing 30 cm, plant spacing 10 cm). At maturity, three plants were randomly collected from each row, and the harvested wheat grains were placed at 105 °C for 30 min and then dried at 80 °C to constant weight. After weighing the dry weight, grind and sieve, weigh 0.1-0.2 g of sample and add 5 mL of mixed acid (HClO4 and HNO3 with a volume ratio of 4:1) to digest for 12 h, then heat and digest at 220 °C for 4-5 h, and stop when the solution is colorless and transparent and there is still 1.00 mL. After the liquid cools naturally, it is fixed to volume with 1% HNO3 and filtered. Finally, inductively coupled plasma mass spectrometry (ICP-MS; Nexlon 2000, RerkinElmer, USA) was used to determine the contents of copper and magnesium.

[0042] (3) 55K SNP chip analysis

[0043] a) DNA extraction: The CTAB method was used to extract DNA from the parental lines “Ailanmai”, “LM001” and the recombinant inbred line population.

[0044] b) The extracted DNA was quality tested using an ultra-micro spectrophotometer, and qualified samples were sent to the company for genotype analysis. In this study, the genotype analysis of the parent-mapping population was completed using the wheat 55K SNP chip developed in collaboration between Beijing Bio-Jingdian Biotechnology Co., Ltd. and Jia Jizeng's research group.

[0045] c) Construction of genetic linkage map: Based on the 55K SNP chip data, a genetic linkage map was constructed using JoinMap4.0. Combined with the maximum root length phenotype data of the population, the Inclusive Composite Interval Mapping-ADD (ICIM-ADD) method in QTL IciMapping 4.2 was used to detect QTLs with a threshold of LOD ≥ 2.5, using BLUP (best linear unbiased prediction) values ​​of grain copper and magnesium content at 5 ecological points and 5 ecological points. The QTL QGrCuc.sau-4A for copper content and QTl QGrMgc.sau-4A for magnesium content in wheat grains were located, and the positions of QGrCuc.sau-4A and QGrMgc.sau-4A and the genetic distance between molecular markers were calculated.

[0046] d) Comparison of copper and magnesium content loci in grains and acquisition of SNP molecular markers

[0047] In order to further densify the map and obtain SNP molecular markers that are closely linked to QGrCuc.sau-4A and QGrMgc.sau-4A, the flanking markers were physically located using the 55K SNP chip data positioning results and the genes located within the interval were screened. The genes were further sequenced and polymorphic sites were mined to develop efficient KASP molecular markers.

[0048] At present, the Durum Wheat v1.0 reference genome sequence is available. However, because there may be chromosome structural variations in the process of wheat evolution, the gene sequence structure and polymorphic sites of different wheat genotypes may be quite different. To obtain the gene sequence of a specific wheat genotype efficiently, quickly and at low cost, the simplest method is homologous sequence cloning. Because tetraploid wheat has two partially homologous chromosome sets, it is very difficult to separate and obtain sequences specific to one of the homologous chromosomes, and primers specific to a certain chromosome set need to be designed. On the basis of mastering comparative genomics and bioinformatics techniques, the sequence of reference genomes such as tetraploid wild emmer wheat and Durum wheat is intercepted, aligned and analyzed to obtain polymorphic sites specific to a certain chromosome, so as to design specific primers for amplifying the target region. After the primers are designed, comparative genomics techniques need to be used to further analyze the primer specificity, annealing temperature, amplification length, etc. to determine the availability of the primers. In summary, although KASP marker technology has been widely used in diploid species, it is not easy to obtain efficient KASP markers in tetraploid wheat.

[0049] The present invention designed 5 pairs of KASP primers through multiple cloning and sequencing, primer design and amplification, as shown in Table 1 below.

[0050] Table 1 KASP primer sequences

[0051]

[0052] e) Conduct analysis

[0053] The five pairs of KASP primers in Table 1 above were used to perform labeling detection on the recombinant inbred line population obtained in step (2), the female parent "Ailanmai" and the male parent "LM001". The specific method was to extract DNA from each strain; use it as a template and perform fluorescence quantitative PCR using specific primers of KASP.

[0054] The reaction system of fluorescence quantitative PCR was as follows: 5 μL Master Mix, 5 ng template DNA, 1.4 μL mixed primers (the mixed primers were prepared by mixing F-FAM primers, F-HEX primers and R primers with a concentration of 10 ng / μL in each group of KASP primer pairs in volumes of 120 μL, 120 μL and 300 μL, respectively, and adding 460 μL ddH2O), ddH2O was added to a total of 10 μL, and 3 independent blank control groups were added with ddH2O instead of template DNA.

[0055] The reaction procedure of fluorescence quantitative PCR was as follows: pre-denaturation at 94 °C for 15 min; denaturation at 94 °C for 20 s, annealing / extension at 61 °C for 60 s, for a total of 10 cycles; denaturation at 94 °C for 20 s, annealing / extension at 55 °C for 60 s, for a total of 26 cycles; fluorescence reading was performed after completion.

[0056] The experimental results and the phenotypic identification results obtained in step (2) were analyzed, and the data were fitted using Origin software. Among them, only KASP-4A-sau1 was significantly correlated with the grain copper content QTL QGrCuc.sau-4A and the grain magnesium content QTL QGrMgc.sau-4A (such as Figure 1 As shown in the figure, the other four primer pairs could not be tightly linked to the target traits, so the SNP molecular marker KASP-4A-sau1 that was tightly linked to the grain copper content QTL QGrCuc.sau-4A and the magnesium content QTL QGrMgc.sau-4A was finally obtained.

[0057] The experimental results of labeling detection with KASP-4A-sau1 are as follows Figure 2 As shown, the plants whose test results showed consistent HEX (square) fluorescence with the father parent "LM001" were strains with higher grain copper and magnesium contents, and the plants whose test results showed consistent FAM (circle) fluorescence with the mother parent "Ailanmai" were strains with lower grain copper and magnesium contents.

[0058] Example 2: Application of SNP molecular marker KASP-4A-sau1 in selecting QTL QGrCuc.sau-4A for controlling grain copper content and QTL QGrMgc.sau-4A for controlling magnesium content

[0059] (1) Recombinant inbred lines were constructed using tetraploid wheat "PI 503554" with lower grain copper and magnesium contents as the male parent and tetraploid wheat "LM001" with higher grain copper and magnesium contents as the female parent. 100 lines were randomly selected from the offspring lines.

[0060] (2) The 100 strains obtained were subjected to KASP-4A-sau1 marker detection. The specific method was as follows: DNA of the 100 strains was extracted respectively, used as a template, and fluorescent PCR was performed using specific primers of the molecular marker KASP-4A-sau1.

[0061] The reaction system of the above fluorescent PCR is: 5 μL Master Mix, 5 ng template DNA, 1.4 μL mixed primers (the mixed primers are prepared by mixing the primers shown in the sequences of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in volumes of 120 μL, 120 μL and 300 μL, respectively, and the concentration of the above primers is 10 ng / μL, and 460 μL ddH2O is added), ddH2O is added to a total amount of 10 μL, and 3 independent blank control groups in which ddH2O is used instead of template DNA are added.

[0062] The reaction program of fluorescence PCR was as follows: pre-denaturation at 94 °C for 15 min; denaturation at 94 °C for 20 s, annealing / extension at 61 °C for 60 s, for a total of 10 cycles; denaturation at 94 °C for 20 s, annealing / extension at 55 °C for 60 s, for a total of 26 cycles; fluorescence reading was performed after completion.

[0063] Fluorescence readings are as follows Figure 3 As shown, the plant genotype detected with HEX (square) fluorescence consistent with "LM001" is recorded as A, which is a strain with higher grain copper and magnesium content, the plant genotype that shows FAM (circle) fluorescence like "PI 503554" is recorded as B, which is a strain with lower grain copper and magnesium content, the triangular fluorescence is a heterozygous strain, and the black diamond fluorescence is a blank control. The field phenotypic values ​​of grain copper and magnesium content were measured using the method of step (2) in Example 1, and the genotypes of each strain and the field phenotypic values ​​of grain copper and magnesium content are shown in Table 2 below.

[0064] Table 2 Correspondence between KASP-4A-sau1 genotype and phenotype

[0065]

[0066] According to the average copper and magnesium content test results and the KASP-4A-sau1 genotype test results in Table 2 above, the present invention can quickly and accurately detect the grain copper and magnesium content QTL on wheat chromosome 4A through the SNP molecular marker KASP-4A-sau1, and can accurately screen out strains with higher and lower grain copper and magnesium contents, thereby improving the efficiency of breeding work and facilitating molecular assisted breeding of wheat.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A SNP molecular marker simultaneously linked to QTLs for copper and magnesium content in wheat, characterized in that: The SNP molecular marker polymorphism is A / G, which is co-localized with the wheat copper content QTL QGrCuc.sau-4A and the magnesium content QTL QGrMgc.sau-4A on the short arm of chromosome 4A of the wheat genome. The SNP molecular marker is amplified by the primer combination shown in SEQ ID NO.1, SEQ ID NO.2 and SEQID NO.

3.

2. A primer combination for amplifying the SNP molecular marker according to claim 1, characterized in that: The primer combination includes two upstream primers as shown in SEQ ID NO.1 and SEQ ID NO.2 and one downstream primer as shown in SEQ ID NO.

3.

3. Use of the primer combination according to claim 2 in preparing a chip for identifying wheat genes or gene fragments thereof.

4. Use of the primer combination according to claim 2 in preparing a kit for identifying wheat genes or gene fragments thereof.

5. A chip for identifying wheat genes or gene fragments thereof, characterized in that: Comprising the primer combination described in claim 2.

6. A kit for identifying wheat genes or gene fragments thereof, characterized in that: Comprising the primer combination described in claim 2.

7. Use of the SNP molecular marker according to claim 1 or the primer combination according to claim 2 or the chip according to claim 5 or the kit according to claim 6 in wheat molecular genetic breeding or cultivation of transgenic wheat or improvement of wheat germplasm resources or screening of wheat lines / variety or genetic analysis and positioning of wheat copper and magnesium content genes.

8. A method for screening wheat lines containing both copper content QTL QGrCuc.sau-4A and magnesium content QTL QGrMgc.sau-4A, characterized in that: The following steps are involved: The genomic DNA of the plant sample to be tested is used as a template, and the primer combination described in claim 2 is used for fluorescence quantitative PCR amplification. Then, the amplification result is used for genotyping, and the plant that can read the fluorescent group marked by the sequence such as SEQ ID NO.2 is identified as a wheat line containing both the copper content QTL QGrCuc.sau-4A and the magnesium content QTL QGrMgc.sau-4A.

9. The method for screening wheat lines containing both the copper content QTL QGrCuc.sau-4A and the magnesium content QTL QGrMgc.sau-4A according to claim 8, characterized in that: The reaction system of the fluorescent quantitative PCR amplification is: 5 μL Master Mix, 5 ng template DNA, 1.4 μL mixed primers, and ddH2O added to a total volume of 10 μL; The mixed primer consists of an upstream primer solution shown in SEQ ID NO.1, an upstream primer solution shown in SEQ ID NO.2, a downstream primer solution shown in SEQ ID NO.3 and ddH2O; The volume ratio of the upstream primer solution shown in SEQ ID NO.1, the upstream primer solution shown in SEQ ID NO.2, the downstream primer solution shown in SEQ ID NO.3 and ddH2O is (100-150): (100-150): (250-350): (400-500); The concentration of the upstream primer solution shown in SEQ ID NO.1 is 8-12 ng / μL, the concentration of the upstream primer solution shown in SEQ ID NO.2 is 8-12 ng / μL, and the concentration of the downstream primer solution shown in SEQ ID NO.3 is 8-12 ng / μL.

10. The method for screening wheat lines containing both the copper content QTL QGrCuc.sau-4A and the magnesium content QTL QGrMgc.sau-4A according to claim 8 or 9, characterized in that: The reaction procedure of the fluorescent quantitative PCR amplification is: first pre-denaturation at 94°C for 15 minutes, then denaturation at 94°C for 20 seconds, annealing / extension at 61°C for 60 seconds, for a total of 10 cycles; finally denaturation at 94°C for 20 seconds, annealing / extension at 55°C for 60 seconds, for a total of 26 cycles.

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