An indel molecular marker related to cadmium accumulation in wheat kernels and application thereof

Abstract

The application discloses a wheat grain cadmium accumulation related Indel molecular marker and application thereof, and belongs to the technical field of molecular markers. The Indel molecular marker is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 1. The application compares TaHMTP functional genes of different wheat germplasm with different cadmium contents, determines a specific 188bp deletion polymorphism in a DNA sequence, and applies the 188bp deletion polymorphism to molecular marker assisted selection of wheat grain cadmium content. The application can judge wheat grain cadmium accumulation germplasm by detecting whether the 188bp fragment is contained in the genome of different wheat varieties, and can quickly screen a new wheat grain cadmium low accumulation variety and improve the breeding efficiency of the new wheat grain cadmium low accumulation variety.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to an Indel molecular marker related to cadmium accumulation in wheat grains and its applications. Background Technology

[0002] Cadmium (Cd) is one of the most toxic heavy metals known to organisms and is most prevalent in agricultural pollution. Cadmium in soil primarily originates from natural factors such as mineral deposits, rocks, and atmospheric deposition, as well as human factors such as the rampant discharge of industrial waste ("three wastes") from mining, smelting, and agricultural production. Human activities are considered the main cause of cadmium pollution. Cadmium has a long half-life and is not easily decomposed by microorganisms, leading to irreversible accumulation once it enters the soil.

[0003] Cadmium is highly mobile in soil, easily absorbed by crops and transferred to the above-ground parts, affecting not only crop quality and yield but also entering the human body through the food chain, seriously endangering human health. my country faces a severe soil cadmium pollution problem. The "Joint Report on the Status Quo of Soil Pollution in China" released by the Ministry of Ecology and Environment and the Ministry of Land and Resources in 2014 showed that 82.4% of the soil samples identified as polluted were caused by metals and metalloids, with cadmium (7%) ranking highest among heavy metals. Soil cadmium pollution has become one of the major obstacles restricting my country's food security and sustainable agricultural development. For large-scale, moderately to lightly cadmium-contaminated farmland, the most economical and effective approach is to cultivate and plant varieties with low cadmium accumulation in edible parts.

[0004] Reducing cadmium accumulation in the edible parts of crops has become an important scientific task for the sustainable and efficient use of land resources and for ensuring the quality and safety of agricultural products. Wheat is the world's most important food crop and one of the most widely planted. With economic development and population growth, the demand for wheat is constantly increasing, and excessive cadmium in grains has become one of the biggest threats to wheat production safety. Studies have shown that there are significant differences in cadmium accumulation among different wheat varieties, and the cadmium content in grains mainly depends on the cadmium content in roots and aboveground parts. Cadmium migration in the aboveground parts controls cadmium accumulation in wheat grains (Effects of node restriction on cadmium accumulation in eight Chinese wheat (Triticum turgidum) cultivars. Science of the Total Environment, 2020, 725.). Yang Yumin et al. found that different wheat varieties had significant differences in grain cadmium content and cadmium migration accumulation coefficient, and there were obvious genotypic differences. Using genotypic differences, they screened out 5 wheat varieties with low grain cadmium content and relatively stable performance (Cadmium accumulation and growth response of wheat varieties promoted in Sichuan under cadmium stress [J]. Southwest China Journal of Agricultural Sciences, 2018, 31(9):1796-1801.).

[0005] Therefore, rapid, accurate, and simple identification of cadmium accumulation characteristics in wheat grains is of great significance for screening and breeding new wheat varieties with low cadmium accumulation. Summary of the Invention

[0006] The purpose of this invention is to provide an Indel molecular marker associated with cadmium accumulation in wheat grains, which can be used to identify wheat germplasm with high and low cadmium accumulation, thereby accelerating the selection and breeding process of wheat varieties with low cadmium accumulation in the field of wheat genetics and breeding.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides an Indel molecular marker associated with cadmium accumulation in wheat grains, wherein the Indel molecular marker is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO.1.

[0009] The Indel molecular marker consists of 188 nucleotides and is located on wheat chromosome 2B. This invention demonstrates that two haplotypes exist in wheat germplasm with different grain cadmium contents. In low-cadmium-accumulating varieties, a 188 bp fragment is present in the TaHMTP (Heavy metaltransport / detoxification superfamily protein) gene, while this fragment is absent in high-cadmium-accumulating varieties. Therefore, detecting the presence of this fragment in wheat genomic DNA can aid in the screening of wheat germplasm with low cadmium accumulation.

[0010] This invention provides the application of the Indel molecular marker in identifying wheat grain cadmium-accumulating varieties or assisting in the screening of wheat grain cadmium-low accumulating varieties. The application includes: detecting whether the Indel molecular marker is present in the genomic DNA of the wheat to be tested using molecular biological methods; if the Indel molecular marker is present, the wheat to be tested is determined to be a low-cadmium-accumulating variety, otherwise it is determined to be a high-cadmium-accumulating variety.

[0011] The wheat grain cadmium-low accumulation varieties have a cadmium content of less than 0.050 mg / kg, while the wheat grain cadmium-high accumulation varieties have a cadmium content of ≥0.050 mg / kg.

[0012] Furthermore, the application includes: using PCR technology to detect the wheat genomic DNA to be tested, wherein the PCR reaction system includes PCR primers for amplifying wheat genomic DNA fragments containing the Indel molecular marker.

[0013] Preferably, the PCR primers include an upstream primer and a downstream primer, wherein the upstream primer is 5'-GTGGCAGTCTGGAATGAGCA-3' and the downstream primer is 5'-TCTGAGCAAAGAATAGGCGCA-3'.

[0014] The above primer pairs can be used to perform PCR amplification on different wheat genotypes to screen wheat germplasm resources with different grain cadmium contents. Specifically, when the wheat to be tested is a low-cadmium-accumulating variety, the PCR product obtained by amplification using the above primers is 1521 bp, and the nucleotide sequence is shown in SEQ ID NO.2, which contains a 188 bp Indel molecular marker (Indel+188), with the Indel start site located after 419 bp of the amplified fragment. When the wheat to be tested is a high-cadmium-accumulating variety, the PCR product obtained by amplification using the above primers is 1345 bp, and the nucleotide sequence is shown in SEQ ID NO.3, which lacks the 188 bp Indel molecular marker (Indel-188).

[0015] This invention also provides a method for identifying or assisting in the identification of wheat grain cadmium-accumulating varieties, comprising the following steps:

[0016] (1) Using the wheat genomic DNA to be tested as a template, PCR amplification was performed using the above PCR primers to obtain PCR products;

[0017] (2) Wheat varieties are identified based on whether the PCR product contains the Indel molecular marker with a nucleotide sequence as shown in SEQ ID NO.1;

[0018] The cadmium accumulation in the wheat grains of the test wheat whose PCR product did not contain the Indel molecular marker was higher than that in the test wheat whose PCR product contained the Indel molecular marker.

[0019] The Indel molecular marker can be detected in various tissues and developmental stages of wheat, thus allowing for screening during the wheat seedling stage and accelerating the selection and breeding process. Preferably, in step (1), genomic DNA is extracted from leaves of the wheat seedlings to be tested.

[0020] Preferably, the PCR amplification reaction system is as follows: 10 μL of 2×Rapid Taq Master Mix, 1 μL of 10 μmol / L Primer, 0.04-0.4 μg of template DNA, and 20 μL of sterile water.

[0021] Preferably, the PCR reaction program includes: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0022] In step (2), the position and size of the amplified product bands are determined by agarose gel electrophoresis. If the amplified product contains a 188bp sequence, the wheat to be tested is a low-cadmium-accumulation material in the grains; if the amplified product does not contain a 188bp sequence, the wheat to be tested is a high-cadmium-accumulation material in the grains.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention compares the TaHMTP functional gene of wheat germplasm with different grain cadmium content and identifies a unique 188bp deletion polymorphism in its DNA sequence. This polymorphism is used as a molecular marker for marker-assisted selection of wheat grain cadmium content. By detecting whether the genome of different wheat varieties contains this 188bp fragment, the germplasm of wheat grain cadmium accumulation can be judged, and new wheat varieties with low grain cadmium accumulation can be quickly screened, thereby improving the breeding efficiency of new wheat varieties with low grain cadmium accumulation. Attached Figure Description

[0025] Figure 1Electrophoresis diagrams of PCR amplification products from eight wheat germplasms with different cadmium contents in their grains.

[0026] Figure 2 The average cadmium content in wheat grains of two haplotypes.

[0027] Figure 3 The cadmium content of two haplotypes in grains from 75 wheat germplasms was determined. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0030] Example 1

[0031] (1) Test materials

[0032] Using eight core germplasm accessions as materials, the seeds were planted in Ningbo, Zhejiang Province in 2018-2019. The seeds were harvested at maturity, dried, and then stored.

[0033] (2) Determination of cadmium content in grains

[0034] The seeds were ground into powder, and 0.3g was weighed and placed in a nitration tube with nitric acid. Cadmium was extracted from the seeds using microwave digestion, and the cadmium content in the extract was determined by ICP-MS. The results are shown in Table 1.

[0035] Table 1

[0036]

[0037]

[0038] (3) Determination of Indel molecular markers

[0039] In our previous study, we identified a QTL on chromosome 2B that was significantly associated with cadmium content in wheat grains through genome-wide association analysis. Within this QTL region, combined with gene annotation, we screened for a gene related to heavy metal transport and detoxification, TaHMTP (HEAVY METAL TRANSPORT / DETOXIFICATION SUPERFAMILY PROTEIN), with GeneID TraesCS2B02G023300 and Location 2B: 10867999-10869126.

[0040] The whole-genome DNA sequence of this gene was amplified in different germplasms. The upstream primer F was 5'-GTGGCAGTCTGGAATGAGCA-3'; the downstream primer R was 5'-TCTGAGCAAAGAATAGGCGCA-3'. The method is as follows:

[0041] 1) DNA extraction

[0042] Place approximately 2 cm long wheat leaves into a 1.5 ml centrifuge tube containing grinding beads, add 500 μL of 1.5×CTAB, set the grinder program (frequency: 50 Hz; time: 2 min), and grind. After grinding, place the tube in a 56℃ water bath and heat for 20 min. After the water bath, add 500 μL of chloroform and place the tube in a shaker, setting the speed to 60 rpm for 30 min. Then place the centrifuge tube in a centrifuge and centrifuge at 8000 rpm for 10 min. Transfer 400 μL of the supernatant to a new centrifuge tube, add 800 μL of cooled anhydrous ethanol, and place the tube at -20℃ for 20 min. After cooling, centrifuge at 12000 rpm for 10 min, discard the supernatant, add 500 μL of 75% ethanol, and centrifuge again at 12000 rpm for 5 min, discarding the supernatant. Finally, place the tube in a clean bench to air dry or let it air dry naturally, then dissolve it in 30-100 μL of ddH2O for later use.

[0043] 2) PCR reaction

[0044] The PCR amplification reaction system was as follows: 10 μL of 2×Rapid Taq Master Mix (Vazyme), 1 μL of 10 μmol / L Primer, 1 μL of template DNA (0.04-0.4 μg template in a 20 μL system), 7 μL of sterile water, and a total reaction volume of 20 μL.

[0045] The PCR reaction was performed on a PCR instrument. The reaction program included: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0046] After the reaction, agarose gel electrophoresis was performed, and the results are as follows: Figure 1 As shown.

[0047] Whole-genome DNA amplification of eight wheat germplasm resources revealed differences in the electrophoretic bands, with amplified sizes of 1521 bp and 1345 bp, respectively. Sequencing yielded nucleotide sequences as shown in SEQ ID NO.2 and SEQ ID NO.3, respectively. Sequencing results showed two haplotypes of this sequence in cadmium-accumulating varieties. A 188 bp fragment, as shown in SEQ ID NO.1, was present in low-cadmium-accumulating varieties, with the start site located after 419 bp of the amplified fragment SEQ ID NO.2. This fragment was absent in high-cadmium-accumulating varieties, exhibiting not only the large 188 bp deletion but also other minor base differences.

[0048] Combine the tabular data to create a bar chart, such as Figure 2 As shown, the average cadmium content in the kernels of the low-cadmium-accumulating variety (Indel+188) was 0.017 mg / kg. -1 The average cadmium content in the grains of the high-cadmium-accumulating variety (Indel-188) was 0.095 mg / kg. -1 .

[0049] Example 2

[0050] (1) Test materials

[0051] Using 75 core germplasm accessions as materials, the seeds were planted in Ningbo, Zhejiang Province in 2018-2019. The seeds were harvested at maturity, dried, and then stored.

[0052] (2) Determination of cadmium content in grains

[0053] The seeds were ground into powder, and cadmium was extracted from the seeds by microwave digestion. The cadmium content in the extract was determined by ICP-MS, and the results are shown in Table 2.

[0054] (3) Indel molecular marker detection

[0055] Genomic DNA was extracted from 75 wheat plants and amplified by PCR using the aforementioned primer pairs. The band size was detected by agarose gel electrophoresis to determine whether there was a 188bp insertion. The results are shown in Table 2.

[0056] Table 2

[0057]

[0058]

[0059]

[0060] Based on phenotypic data, the cadmium content in the grains of the Indel-188 type was significantly higher than that in the Indel+188 type, as shown in the following results. Figure 3 As shown.

[0061] We expanded the population and amplified the DNA sequences of 75 wheat germplasm resources. Using SPSS, we performed Pearson correlation analysis on two variables: grain cadmium content and the presence of a 188bp fragment. The results showed a correlation coefficient of 0.81, which was significantly correlated with grain Cd content at the 0.01 level. This indicates that the insertion of this fragment is correlated with grain cadmium content and can be used as a molecular marker to screen for new varieties with low cadmium accumulation.

[0062] The results above demonstrate that this Indel molecular marker can be applied to marker-assisted selection of cadmium content in wheat grains.

Claims

1. An Indel molecular marker associated with cadmium accumulation in wheat grains, characterized in that, The Indel molecular marker is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO.

1.

2. The application of the Indel molecular marker as described in claim 1 in identifying wheat grain cadmium-accumulating varieties or assisting in the screening of wheat grain cadmium-low accumulation varieties, characterized in that, The application includes: detecting whether the wheat genomic DNA to be tested contains the Indel molecular marker using molecular biology methods; if the Indel molecular marker is present, the wheat to be tested is determined to be a low-cadmium-accumulating variety in grains, otherwise it is determined to be a high-cadmium-accumulating variety in grains.

3. The application as described in claim 2, characterized in that, The application includes: using PCR technology to detect wheat genomic DNA to be tested, wherein the PCR reaction system includes PCR primers for amplifying wheat genomic DNA fragments containing the Indel molecular marker.

4. The application as described in claim 3, characterized in that, The PCR primers include an upstream primer and a downstream primer. The upstream primer is 5'-GTGGCAGTCTGGAATGAGCA-3', and the downstream primer is 5'-TCTGAGCAAAGAATAGGCGCA-3'.

5. A method for identifying or assisting in the identification of wheat grain cadmium-accumulating varieties, characterized in that, Includes the following steps: (1) Using the wheat genomic DNA to be tested as a template, PCR amplification is performed using the PCR primers described in claim 3 or 4 to obtain PCR products; (2) Wheat varieties are identified based on whether the PCR product contains the Indel molecular marker with a nucleotide sequence as shown in SEQ ID NO.1; The cadmium accumulation in the wheat grains of the test wheat whose PCR product did not contain the Indel molecular marker was higher than that in the test wheat whose PCR product contained the Indel molecular marker.

6. The method as described in claim 5, characterized in that, In step (1), genomic DNA is extracted from wheat seedling leaves.

7. The method as described in claim 5, characterized in that, The PCR amplification reaction system was as follows: 10 μL of 2×Rapid Taq MasterMix, 1 μL of 10 μmol / L Primer, 0.04-0.4 μg of template DNA, and 20 μL of sterile water.

8. The method as described in claim 5, characterized in that, The PCR reaction procedure included: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min, and storage at 4℃.

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