An InDel molecular marker sequence related to drought resistance in wheat, its detection method, and its application.

Abstract

This invention provides an InDel molecular marker sequence related to drought resistance in wheat, along with its detection method and application, belonging to the field of molecular marker technology. This invention provides a polymorphic site that forms different drought resistance characteristics in wheat. This polymorphic site contains only two haplotypes: a 724bp long sequence insertion (as shown in SEQ ID No. 2) between 245-246bp of the fragment shown in SEQ ID No. 1, indicating haplotype A homozygous; and a haplotype B homozygous, indicating haplotype B, without the 724bp long sequence insertion at the same site. Wheat varieties identified as homozygous for haplotype A exhibit higher drought resistance than those identified as homozygous for haplotype B. Therefore, in marker-assisted selection breeding of wheat, detecting the polymorphic site can identify wheat varieties with relatively high drought resistance.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to an InDel molecular marker sequence related to wheat drought resistance, its detection method, and its application. Background Technology

[0002] Plants grow and develop in complex and ever-changing environments, often subjected to abiotic stresses. Drought is a major abiotic factor affecting and limiting plant growth and development, and can even lead to plant death. Therefore, breeding drought-resistant crop varieties has always been one of the main goals of agricultural science and technology research.

[0003] wheat( Triticum aestivum Wheat (L.) is a major source of carbohydrates and protein. Therefore, there is an urgent need to find effective breeding methods to increase wheat yield. Although wheat planting area and yield have increased, abiotic stresses such as drought, high temperature, and high salinity can all affect wheat yield. Research shows that the key to genetic improvement of crop drought resistance is the cloning and utilization of superior drought-resistant genes. Therefore, the discovery of drought-resistant genes in wheat is of great significance for breeding drought-resistant wheat varieties and increasing wheat yield.

[0004] DNA methylation is a chemical modification process in which methyl groups (-CH3) are added to certain regions of the DNA genomic genome. It is one of the earliest discovered and most thoroughly studied epigenetic regulatory mechanisms. Methylation mainly regulates gene expression by altering the chemical structure of the DNA double helix and the structure of chromatin. The core mechanism is to inhibit the binding of transcription factors to the DNA sequence, thereby hindering gene transcription and reducing its expression level. Therefore, molecular markers of wheat DNA methylation, especially genotyping, are key to solving the drought resistance of wheat varieties. Summary of the Invention

[0005] This invention provides an InDel molecular marker sequence related to wheat drought resistance, a detection method, and its application. The InDel molecular marker can be used to determine wheat haplotypes, which is helpful for wheat breeding.

[0006] This invention provides an InDel molecular marker sequence related to drought resistance in wheat, with an InDel molecular marker site between 245-246 bp of the sequence shown in SEQ ID No. 1, and the nucleotide sequence of the InDel fragment is shown in SEQ ID No. 2.

[0007] The present invention also provides primer pairs for amplifying the above-mentioned InDel molecular marker sequence, including an upstream primer with nucleotide sequences as shown in SEQ ID No. 3 and a downstream primer as shown in SEQ ID No. 4.

[0008] The present invention also provides a kit for identifying or assisting in the identification of wheat drought resistance, comprising the above-mentioned primer pairs.

[0009] The present invention also provides the application of the above-mentioned InDel molecular marker sequence, the above-mentioned primer pair, or the above-mentioned kit in the identification or auxiliary identification of wheat drought resistance.

[0010] The present invention also provides a method for identifying or assisting in the identification of wheat genotypes based on the above-mentioned InDel molecular marker sequence, comprising using the genomic DNA of the wheat to be tested as a template to amplify the above-mentioned InDel molecular marker sequence, and determining that the amplification product contains the sequence described in SEQ ID No. 2 as drought-resistant haplotype A, and determining that the sequence shown in SEQ ID No. 2 is missing as non-drought-resistant haplotype B.

[0011] In a preferred embodiment of the present invention, the amplification product of the drought-resistant monomer type A is 1352bp; and the amplification product of the non-drought-resistant monomer type B is 628bp.

[0012] In a preferred embodiment of the present invention, the amplification program includes: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min, for 38 cycles; and further extension at 72°C for 5 min.

[0013] This invention also provides the application of the above-mentioned InDel molecular marker sequence, primer pair, or kit in improving wheat drought resistance.

[0014] This invention also provides the application of the above-mentioned InDel molecular marker sequence, primer pair, or kit in the breeding and / or assisted breeding of drought-resistant wheat varieties.

[0015] The present invention also provides a method for screening drought-resistant wheat varieties, comprising using the genomic DNA of the wheat to be tested as a template to amplify the above-mentioned InDel molecular marker sequence, selecting drought-resistant haplotype A containing the sequence described in SEQ ID No. 2 in the amplification product, and discarding non-drought-resistant haplotype B that lacks the sequence shown in SEQ ID No. 2 in the amplification product.

[0016] Beneficial effects: This invention provides genes related to drought resistance from a natural variation population of 200 wheat varieties. TaDi19-1Genetic variation analysis identified a polymorphic locus (InDel-1478) contributing to different drought resistance traits in wheat. This polymorphic locus exhibits only two haplotypes: Genotype A, which contains a 724 bp sequence insertion (as shown in SEQ ID No. 2) between 245-246 bp of the fragment represented by SEQ ID No. 1; and genotype B, which does not contain the 724 bp sequence insertion (as shown in SEQ ID No. 2). Wheat varieties identified as homozygous for haplotype A exhibit higher drought resistance than those identified as homozygous for haplotype B. Therefore, in marker-assisted selection breeding of wheat, detecting this polymorphic locus can identify wheat varieties with relatively higher drought resistance. This invention provides a novel method for marker-assisted selection breeding of wheat, which is of significant importance in the development and research of drought-resistant wheat varieties. Attached Figure Description

[0017] Figure 1 The results of qRT-PCR for T3 generation overexpressing wheat lines;

[0018] Figure 2 The phenotype of T3 generation overexpressing wheat lines after drought treatment and rehydration for 3 days;

[0019] Figure 3 The survival rate of T3 generation overexpressing wheat lines after drought treatment and rehydration for 3 days is statistically shown.

[0020] Figure 4 Infrared thermal imaging results of T3 generation overexpressing wheat plants under normal growth and drought stress conditions;

[0021] Figure 5 The leaf temperature of the transgenic wheat plants under normal growth and drought stress conditions in Example 3;

[0022] Figure 6 The results of target editing for T3 generation wheat lines;

[0023] Figure 7 Phenotypes of T3 generation wheat lines after drought treatment and rehydration for 3 days;

[0024] Figure 8 The survival rate of T3 generation edited wheat lines after drought treatment and rehydration for 3 days is statistically analyzed.

[0025] Figure 9 Infrared thermal imaging results of T3 generation overexpressing wheat plants under normal growth and drought stress conditions;

[0026] Figure 10 Leaf temperature of transgenic wheat plants under normal growth and drought stress conditions in Example 3.

[0027] Figure 11 The 724bp sequence difference between the two monomer types of this invention in drought-resistant and drought-sensitive wheat;

[0028] Figure 12 The results of PCR amplification of the molecular marker InDel-1478 in the two monomeric forms of this invention;

[0029] Figure 13 For 200 wheat varieties TaDi19-1 Gene nucleotide polymorphism analysis;

[0030] Figure 14 for TaDi19-1 Results of LUC activity analysis in tobacco leaves using two monomeric promoters;

[0031] Figure 15 for TaDi19-1 GUS staining results of Arabidopsis thaliana with two haplotype promoters;

[0032] Figure 16 for TaDi19-1 Results of McBC-qPCR for DNA methylation of two monomeric sequences;

[0033] Figure 17 for TaDi19-1 BSP results of methylation of monomeric A-sequence DNA;

[0034] Figure 18 for TaDi19-1 qRT-PCR results of two monomeric parental materials;

[0035] Figure 19 for TaDi19-1 qRT-PCR results in the NIL population BC4F2 generation;

[0036] Figure 20 The phenotype of the parental materials after drought treatment and rehydration for 3 days;

[0037] Figure 21 The survival rate of parental materials after drought treatment and rehydration for 3 days is statistically shown.

[0038] Figure 22 Phenotypes of NIL population BC4F2 generation material after drought treatment and rehydration for 3 days;

[0039] Figure 23 The survival rate of NIL population BC4F2 generation material after drought treatment and rehydration for 3 days is statistically analyzed. Detailed Implementation

[0040] This invention provides an InDel molecular marker sequence related to drought resistance in wheat, with an InDel molecular marker site between 245-246 bp of the sequence shown in SEQ ID No. 1, and the nucleotide sequence of the InDel fragment is shown in SEQ ID No. 2.

[0041] This invention analyzes drought-related genes in a natural variation population of 200 wheat varieties. TaDi19-1 Genetic variation analysis identified the polymorphic site (InDel-1478) that contributes to different drought resistances in wheat; this polymorphic site contains only two haplotypes. The gene described in this invention... TaDi19-1 The nucleotide sequence of the protein is shown in SEQ ID No. 5, and the amino acid sequence of the encoded protein TaDi19-1 is shown in SEQ ID No. 6. When this gene is introduced into the wheat genome for overexpression, overexpressing plants are constructed. Compared to wild-type plants, the overexpressing plants exhibit reduced drought resistance. When the encoding gene is knocked out, the resulting mutant plants exhibit enhanced drought resistance compared to wild-type plants. This demonstrates that the protein TaDi19-1 or gene described in this invention... TaDi19-1 Negative regulation of drought resistance in plants.

[0042] Table 1. Results of the survey on natural variation populations and drought resistance of 200 wheat varieties.

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050] The 200 wheat varieties shown in Table 1 have all been published in articles (Chen, B., Liu, Y., Yang, Y., Wang, Q., Li, S., Li, F., et al, (2025). A system genetics analysis uncovers theregulatory variants controlling drought response in wheat. PlantBiotechnology Journal, 23(5), 1565-1584.), and are available to the public from Northwest A&F University.

[0051] This invention, through analysis, discovered genes in wheat genomic DNA related to drought resistance. TaDi19-1 Two drought-related haplotypes exist at a polymorphic site 1478 bases upstream of the start codon ATG, namely InDel-1478. Corresponding to positions 245-246 of the sequence shown in SEQ ID No. 1, two drought-related haplotypes exist at this site: one haplotype A has an insertion of 724 bases (SEQ ID No. 2), which is haplotype A; the other haplotype B has no base insertion at this polymorphic site.

[0052] The present invention also provides primer pairs for amplifying the above-mentioned InDel molecular marker sequence, including an upstream primer with nucleotide sequences as shown in SEQ ID No. 3 and a downstream primer as shown in SEQ ID No. 4.

[0053] Based on the conserved sequences flanking InDel-1478, this invention designed the specific primer pair shown in SEQ ID No. 3 and SEQ ID No. 4.

[0054] When using the specific primer pair for amplification, wheat genomic DNA is used as a template for PCR amplification. The PCR amplification program includes: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 1 min, 38 cycles; and 72℃ final extension for 5 min. If the amplification product size is 1352 bp, the wheat to be tested is homozygous for haplotype A; if the amplification product size is 628 bp, the wheat to be tested is homozygous for haplotype B; if the amplification product size is both 1352 bp and 628 bp, the wheat to be tested is heterozygous for haplotypes A and B.

[0055] The present invention also provides a kit for identifying or assisting in the identification of wheat drought resistance, comprising the above-mentioned primer pairs.

[0056] This invention also provides a kit for identifying or assisting in the identification of wheat drought resistance, comprising the aforementioned specific primer pairs for amplifying the target InDel site. The kit also includes other reagents required for PCR amplification, such as dNTPs, Taq DNA polymerase, and a magnesium-containing 10×Taq Buffer, to ensure successful PCR reaction. In one embodiment, the PCR amplification reagent can be a commercially available Taq PCR premix, such as the 2×Taq PCR Mix (Cat. No. RR001A) provided by Takara. Any equivalent commercially available PCR premix or a combination of the aforementioned individual components can be used for PCR amplification according to experimental requirements.

[0057] The present invention also provides the application of the above-mentioned InDel molecular marker sequence, the above-mentioned primer pair, or the above-mentioned kit in the identification or auxiliary identification of wheat drought resistance.

[0058] In this invention, after the monomeric A-type methylation molecule inserts a 724 bp fragment into the promoter region, the methylation level increases, promoter activity decreases, and transcriptional activity decreases, leading to... TaDi19-1 Expression decreases, thereby enhancing wheat drought resistance; while the monomeric B promoter has higher activity. TaDi19-1 Increased expression levels reduce the plant's drought resistance.

[0059] The present invention also provides a method for identifying or assisting in the identification of wheat genotypes based on the above-mentioned InDel molecular marker sequence, comprising using the genomic DNA of the wheat to be tested as a template to amplify the above-mentioned InDel molecular marker sequence, and determining that the amplification product contains the sequence described in SEQ ID No. 2 as drought-resistant haplotype A, and determining that the sequence shown in SEQ ID No. 2 is missing as non-drought-resistant haplotype B.

[0060] The amplification procedure described in this invention includes: pre-denaturation at 98℃ for 3 min; denaturation at 98℃ for 30 s, annealing at 52℃ for 30 s, extension at 72℃ for 1 min, for 38 cycles; and a final extension at 72℃ for 5 min. After the amplification, the amplification product of the drought-resistant monomer type A is 1352 bp; and the amplification product of the non-drought-resistant monomer type B is 628 bp.

[0061] This invention also provides the application of the above-mentioned InDel molecular marker sequence, primer pair, or kit in improving wheat drought resistance.

[0062] Using the InDel molecular markers described in this invention, drought resistance in wheat can be improved based on genotype and haplotype detection. For example, haplotype A of genotype A has stronger drought resistance, while haplotype B of genotype B has weaker drought resistance.

[0063] This invention also provides the application of the above-mentioned InDel molecular marker sequence, primer pair, or kit in the breeding and / or assisted breeding of drought-resistant wheat varieties.

[0064] The present invention also provides a method for screening drought-resistant wheat varieties, comprising using the genomic DNA of the wheat to be tested as a template to amplify the above-mentioned InDel molecular marker sequence, selecting drought-resistant haplotype A containing the sequence described in SEQ ID No. 2 in the amplification product, and discarding non-drought-resistant haplotype B that lacks the sequence shown in SEQ ID No. 2 in the amplification product.

[0065] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides an InDel molecular marker sequence, detection method, and application related to wheat drought resistance, but these should not be construed as limiting the scope of protection of the present invention.

[0066] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0067] In this embodiment of the invention, the naming method for polymorphic sites is a marker type + / - sequence number; wherein, the marker type includes two types: SNP and InDel, where SNP represents single nucleotide polymorphism and InDel represents insertion or deletion; the sequence number is based on the reference genome DNA of wheat Chinese Spring. TaDi19-1 In the start codon (ATG) of a gene, A is marked as "+1"; for example, SNP418 represents a single nucleotide polymorphism at the 418th base downstream of A in the start codon (ATG); InDel-1478 represents an insertion or deletion of one or more bases downstream of the 1478th base upstream of A in the start codon (ATG).

[0068] In the examples below, the positions of primers or sequences are based on the reference genomic DNA of Chinese Spring wheat. TaDi19-1 In the start codon (ATG) of a gene, the A is denoted as "+1", and the first base upstream of the A in the start codon (ATG) is denoted as "-1".

[0069] The genomic sequence of the TaDi19-1 gene in the reference genomic DNA of Chinese Spring wheat is shown in Sequence Listing Sequence 1. The start codon A is located at position 1722 from the 5' end of Sequence 1.

[0070] The biomaterials used in the following examples are as follows:

[0071] Vector pBUE411: documented in the literature: Liu Y, Chen B, Qin Z, Jiang P, Yang Y, et al. TaFAR5-TaFAR3 module regulates cuticular wax biosynthesis and drought tolerance in wheat. New Phytologist 2025. Available to the public from Northwest A&F University;

[0072] Vector pCAMBIA3301: Documented in the literature: Regulatory changes in TaSNAC8-6A Areas associated with drought tolerance in wheat seedlings. Plant Biotechnol J2019. Available to the public from Northwest A&F University;

[0073] Agrobacterium tumefaciens strain GV3101: described in the literature: Jing Y, Zhang D, Wang X, Tang W, Wang W, et al. (2013) Arabidopsis Chromatin remodeling factor PICKLE interacts with transcription factor HY5 to regulate hypocotyl cell elongation. PlantCell 25: 242-256, available to the public from Northwest A&F University;

[0074] Chinese Spring wheat variety: documented in literature as a regulatory change in TaSNAC8-6A These are associated with drought tolerance in wheat seedlings. PlantBiotechnol J 2019. Available to the public from Northwest A&F University;

[0075] Wheat variety Fielder: documented in the literature: Regulatory changes in TaSNAC8-6A Areas associated with drought tolerance in wheat seedlings. Plant Biotechnol J2019. Available to the public from Northwest A&F University.

[0076] Example 1: Obtaining the protein TaDi19-1 and its encoding gene

[0077] I. Cloning of the protein TaDi19-1 and its encoding gene

[0078] Seeds of the wheat cultivar Chinese Spring were germinated at 25℃ for three days. The germinated seeds were then transferred to nutrient soil or nutrient solution and cultured for two weeks. The entire plant was then quick-frozen in liquid nitrogen, ground, and total RNA was extracted. Reverse transcription was performed to obtain cDNA. Using this cDNA as a template, PCR amplification was performed using the primer pairs shown in SEQ ID No. 7 and SEQ ID No. 8. The amplified product was subjected to agarose gel electrophoresis, and a 684 bp DNA fragment was isolated and purified for sequencing. Its sequence is shown in SEQ ID No. 5 and named the gene. TaDi19-1 The encoded amino acid sequence is shown in SEQ ID No. 6, and the protein is named TaDi19-1.

[0079] II. Overexpressed genes TaDi19-1 Reduce wheat drought resistance

[0080] 1. Construction of recombinant vectors

[0081] The DNA fragment shown in SEQ ID No. 5 was cloned into pCAMBIA3301. BamHI The recombinant vector was obtained by sequencing between the restriction enzyme sites (located downstream of the Ubi promoter) and confirmed to express the TaDi19-1 protein shown in SEQ ID No. 6.

[0082] 2. Obtaining recombinant Agrobacterium tumefaciens

[0083] The recombinant vector was transformed into Agrobacterium tumefaciens strain EHA105 to obtain recombinant Agrobacterium Y containing the recombinant vector.

[0084] The empty vector pCAMBIA3301 was transformed into Agrobacterium tumefaciens strain EHA105 to obtain recombinant Agrobacterium CK containing the empty vector pCAMBIA3301.

[0085] 3. Obtaining wheat overexpression lines

[0086] Recombinant Agrobacterium Y was transformed into the wheat variety Fielder (hereinafter also referred to as wild-type wheat) using Agrobacterium-mediated gene transformation to obtain T0 generation plants, which were then planted in a greenhouse (16h light / 8h dark). T0 generation plants were identified as positive by PCR, and after self-pollination, T1 generation seeds were obtained. T1 generation plants were then identified as positive by PCR, and after self-pollination, T2 generation seeds were obtained. Simultaneously, positive and negative seedlings were randomly selected and subjected to qRT-PCR detection according to step 4 to determine overexpression. TaDi19- 1 The expression level was determined. T2 generation plants were then identified by PCR to obtain positive plants, and T3 generation seeds were obtained after self-pollination.

[0087] The recombinant Agrobacterium CK was transformed into the wheat variety Fielder using the method described above until the T3 generation pCAMBIA3301 wheat line was obtained.

[0088] T0 generation represents the plants that grow from the current generation after transformation; T1 generation represents the seeds produced by self-pollination of T0 generation and the plants that grow from them; T2 generation represents the seeds produced by self-pollination of T1 generation and the plants that grow from them; T3 generation represents the seeds produced by self-pollination of T2 generation and the plants that grow from them.

[0089] The specific steps of the Agrobacterium-mediated gene transformation method described above are as follows:

[0090] Recombinant Agrobacterium Y was inoculated into YEB liquid medium containing 25 mg / L spectinomycin and cultured at 28°C with shaking until OD reached. 600The value was 0.5. Wheat embryos were placed in a 2 mL centrifuge tube filled with preservation solution (10 mL of 10×LS Ajor, 1 mL of 100×LS Minor, 1 mL of 100×Fe-EDTA, 1 mL of 100×Vitamin, 10 mL of Glucose and 0.5 g of MES were dissolved in an appropriate amount of water and then diluted to 1 L with water). The tubes were heat-treated at 46 °C for 3 min and centrifuged at 4 °C and 2000 rpm for 10 min. Add the prepared recombinant Agrobacterium to the treated immature embryos and culture in the dark at 22°C for 3 days. Then transfer to selective medium (dissolve 100 mL of 10×LS Major, 10 mL of 100×LS Minor, 10 mL of 100×Fe-EDTA, 10 mL of 100×Vitamin, 5 mL of 2,4-D, 40 g of Maitose, 0.5 g of Glutamine, 0.75 g of MgCl2·6H2O, 1.95 g of MES and 5 g of Agarose in an appropriate amount of water, and then bring the volume to 1 L with water; sterilize at 121°C for 15 min; then add 10 g / L Ascorbic acid, 50 μL of 100 mM AgNO3 solution and 1 mL of 150 g / L Timentin) and culture in the dark at 28°C for 7-10 days. Screening was performed using different concentrations of glufosinate (0.1–3.0 mg / L). Finally, the samples were transferred to differentiation medium (100 mL of 10×LS Major, 10 mL of 100×LS Minor, 10 mL of 100×Fe-EDTA, 10 mL of 100×Vitamin, 50 mL of 100 mg / L Zeatin solution, 100 μL of 100 mM CuSO4·5H2O solution, 20 g Sucrose, 0.5 g MES, and 3 g Gelrite were dissolved in an appropriate amount of water, and then the volume was adjusted to 1 L; sterilized at 121℃ for 15 min; then 250 μL of 20 g / L PPT and 1 mL of 250 g / L Carbenicillin were added). After differentiation, the samples were transferred to rooting medium (100 mL of 10×LS Major, 10 mL of 100×LS Minor, 10 mL of 100×LS Minor, 10 mL of 100 mM CuSO4·5H2O solution, 20 g Sucrose, 0.5 g MES, and 3 g Gelrite were dissolved in an appropriate amount of water, and then the volume was adjusted to 1 L; sterilized at 121℃ for 15 min; then 250 μL of 20 g / L PPT and 1 mL of 250 g / L Carbenicillin were added). Minor, 10 mL of 100×Fe-EDTA, 10 mL of 100×Vitamin, 2 mL of 100 mg / L IBA solution, 15 g of Sucrose, 0.5 g of MES and 3 g of Gelrite were dissolved in an appropriate amount of water, the pH was adjusted to 5.8, and then the volume was brought up to 1 L with water; sterilized at 121℃ for 15 min; then 250 μL of 20 g / L PPT and 1 mL of 250 g / L Carbenicillin were added and cultured on the substrate. After reaching a certain size, the substrate was transferred to nutrient soil.

[0091] 4. qRT-PCR detection of wheat overexpression

[0092] Take the wild-type wheat obtained in step 3 and T3 generation. TaDi19-1 Total RNA was isolated from wheat lines (OE1-OE3) using the TRIZOL (Biotopped) method. Genomic contamination was then eliminated using the DNAseI (Takara) method, followed by concentration determination using a Nanodrop 1000 (Thermo Scientific product, USA). 5 μg of each RNA sample was run on 0.8% agarose gel. 1 μg of total RNA was used to synthesize cDNAs using recombinant M-MLV reverse transcriptase with 1 μg of Oligo(dT)23 (Promega) as a primer. Specific primers F2 and R2 were used to target the gene. TaDi19-1 The cDNA was quantified by qRT-PCR, with the wheat gene TaActin1 as an internal control.

[0093] The sequences of the primers mentioned above are as follows:

[0094] F2 (SEQ ID No. 9): 5'-CATGCAACATGGGTACTTGTTCAAG-3';

[0095] R2 (SEQ ID No. 10): 5'-GGCATTATTGTTGCTCGACCTATGTC-3';

[0096] FC2 (SEQ ID No. 11): 5'-AAATCTGGCATCACACTTTCTAC-3';

[0097] RC2 (SEQ ID No. 12): 5'-GTCTCAAACATAATCTGGGTCATC-3'.

[0098] The results are as follows Figure 1 As shown, T3 generation conversion TaDi19-1 Target gene in wheat lines OE1-OE3 TaDi19-1 The expression level of was significantly higher than that of wild-type WT.

[0099] 5. Phenotypic analysis of drought resistance in wheat overexpression

[0100] Take T3 as the replacement TaDi19-1Wheat lines (OE1, OE2, OE3) and wild-type wheat (WT) plants were transferred to pots containing 250g of nutrient soil. After 21 days of growth under normal conditions, a drought treatment (i.e., watering was stopped) was applied. After 20-30 days, when phenotypic differences became obvious (OE1, OE2, and OE3 lines showed obvious leaf drying while WT plants showed severe leaf wilting), rehydration was initiated. Three days after rehydration, the survival rate of each line was recorded (plants that showed normal growth and could be harvested were defined as surviving plants, and plants that showed severe drought damage and could not grow or be harvested were defined as dead plants; the survival rate was the percentage of surviving plants in each line out of the total number of plants). The experiment was repeated three times, with at least 45 plants from each line in each replicate, and the average value was used for statistical analysis.

[0101] The results are as follows Figure 2 As shown, it can be seen that the T3 generation is converted TaDi19-1 The survival rate of wheat lines after rehydration is lower than that of wild-type wheat.

[0102] Three days after rehydration, the survival rates of each plant line were calculated as follows: Figure 3 As shown, it can be seen that the T3 generation is converted TaDi19-1 The survival rate of wheat lines after rehydration was 25%–35%, significantly lower than that of wild-type wheat.

[0103] Depend on Figure 4 and Figure 5 It can be seen that under normal growth conditions, T3 generation transformation... TaDi19-1 The leaf temperature of wheat lines was no different from that of wild-type wheat, and under drought stress, the T3 generation transition... TaDi19-1 The leaf temperature of wheat lines was significantly lower than that of wild-type wheat. Therefore TaDi19-1 Genes may play an important role in the process of stomatal closure in response to drought.

[0104] III. Knockout TaDi19-1 Enhance wheat drought resistance

[0105] 1. Construction of recombinant vectors

[0106] The DNA fragments shown in SEQ ID No. 13 and SEQ ID No. 14 were inserted into the basic vector pBUE411. HindIII and AscI The recombinant vector pBUE411-KO was obtained between the restriction enzyme sites.

[0107] 2. Obtaining recombinant Agrobacterium tumefaciens

[0108] The recombinant vector was transformed into Agrobacterium tumefaciens strain EHA105 to obtain recombinant Agrobacterium W containing the recombinant vector pBUE411-KO.

[0109] 3. Obtaining wheat knockout materials

[0110] Recombinant Agrobacterium W was transformed into the wheat variety Fielder using Agrobacterium-mediated gene transformation to obtain T0 generation plants, which were then grown in a greenhouse (16h light / 8h darkness). T0 generation plants were identified as positive by PCR, and after self-pollination, T1 generation seeds were obtained. T1 generation plants were then identified as edited plants by high-throughput sequencing, and after self-pollination, T2 generation seeds were obtained. The target editing status was confirmed by high-throughput sequencing. T2 generation plants were then identified as positive by PCR, and after self-pollination, T3 generation seeds were obtained.

[0111] T0 generation represents the plants that grow from the current generation after transformation; T1 generation represents the seeds produced by self-pollination of T0 generation and the plants that grow from them; T2 generation represents the seeds produced by self-pollination of T1 generation and the plants that grow from them; T3 generation represents the seeds produced by self-pollination of T2 generation and the plants that grow from them.

[0112] The specific steps of the Agrobacterium-mediated gene transformation method described above are as follows:

[0113] Recombinant Agrobacterium W was inoculated into YEB liquid medium containing 25 mg / L spectinomycin and cultured at 28°C with shaking until OD reached. 600 The concentration was 0.5. Wheat embryos were placed in 2 mL centrifuge tubes filled with preservation solution (10 mL of 10×LS Ajur, 1 mL of 100×LS Minor, 1 mL of 100×Fe-EDTA, 1 mL of 100×Vitamin, 10 mL of Glucose, and 0.5 g of MES dissolved in an appropriate amount of water, then diluted to 1 L with water). The embryos were heat-treated at 46℃ for 3 min, followed by centrifugation at 4℃ and 2000 rpm for 10 min. The prepared recombinant Agrobacterium was added to the treated embryos and cultured in the dark at 22℃ for 3 days. The embryos were then transferred to selection medium and cultured in the dark at 28℃ for 7–10 days. Screening was performed using different concentrations of glufosinate (0.1–3.0 mg / L). Finally, the embryos were transferred to differentiation medium, and after differentiation, they were transferred to rooting medium. Once a certain size was reached, the embryos were transplanted into nutrient soil.

[0114] 4. High-throughput sequencing detection of knockout wheat materials

[0115] Take the wild-type wheat and T3 generation obtained in step 3. TaDi19-1 The wheat knockout lines (KO1-KO3) were used to extract total DNA using the CTAB method. 1 μg of total DNA was taken and the target sequence was detected by PCR using specific primers PAM1-F / R and PAM2-F / R. The target editing status was then detected by high-throughput sequencing.

[0116] PAM1-F (SEQ ID No. 15): 5'-ggagtgagtacggtgtgcGGTTTGTGCAAGTCTAAAA-3';

[0117] PAM1-R (SEQ ID No. 16): 5'-gagttggatgctggatggTCCAAAGTTAGAGAAGCTC-3';

[0118] PAM2-F (SEQ ID No. 17): 5'-ggagtgagtacggtgtgcCTGAGCCGGGATCTACG-3';

[0119] PAM2-R (SEQ ID No. 18): 5'-gagttggatgctggatggAGGAGAGTAACAGGAAAGAAGA-3'.

[0120] Knockout material target editing status as follows Figure 6 As shown.

[0121] 5. Phenotypic analysis of drought resistance in knockout wheat materials

[0122] Take T3 generation TaDi19-1 Wheat lines (KO1, KO2, KO3) and wild-type wheat (WT) plants were knocked out and transferred to pots containing 250g of nutrient soil. After 21 days of growth under normal conditions, a drought treatment (i.e., watering was stopped) was applied. After 20-30 days, when phenotypic differences became obvious (WT plants showed obvious leaf drying while KO1, KO2, and KO3 plants showed severe leaf wilting), rehydration was initiated. Three days after rehydration, the survival rate of each line was recorded (plants that showed normal growth and could be harvested were defined as surviving plants, and plants that showed severe drought damage and could not grow or be harvested were defined as dead plants; the survival rate was the percentage of surviving plants in each line out of the total number of plants). The experiment was repeated three times, with at least 45 plants from each line in each replicate. The average value was used for statistical analysis.

[0123] The results are as follows Figure 7 As shown, T3 generation TaDi19-1 The survival rate of knockout wheat lines after rehydration was lower than that of wild-type wheat.

[0124] Three days after rehydration, the survival rates of each plant line were calculated as follows: Figure 8 As shown, T3 generation TaDi19-1 The survival rate of the knockout wheat lines after rehydration was 81-85%, which was significantly higher than that of wild-type wheat.

[0125] Depend on Figure 9 and Figure 10 It can be seen that under normal growth conditions, the T3 generation... TaDi19-1The leaf temperature of the knockout wheat lines was no different from that of wild-type wheat, and the T3 generation under drought stress... TaDi19-1 The leaf temperature of the knockout wheat lines was significantly higher than that of the wild-type wheat. This further proves... TaDi19-1 Genes may play an important role in the process of stomatal closure in response to drought.

[0126] In summary, the protein TaDi19-1 and its encoding gene have the function of regulating plant drought resistance, and knocking out [the gene] in plants [is crucial]. TaDi19-1 Genes can enhance a plant's drought resistance.

[0127] Example 2: Haplotypes and their molecular markers related to wheat drought resistance and their applications

[0128] 1. Haplotypes related to wheat drought resistance

[0129] Genes related to drought resistance in wheat genomic DNA TaDi19-1 There are two drought-related haplotypes at the polymorphic site upstream of the start codon ATG, and the polymorphic site is InDel-1478, which corresponds to positions 245-246 of SEQ ID No. 1.

[0130] 2. Identification of the monomeric molecular marker InDel-1748 and its specific primers

[0131] The 200 wheat varieties shown in Table 1 TaDi19-1 The upstream sequence of ATG was amplified, sequenced, and aligned, and the results are as follows: Figure 11 As shown, in TaDi19-1 At position 1478 upstream of the start codon ATG, a 724 bp DNA fragment is inserted in haplotype A homozygous wheat varieties, while no DNA fragment is inserted in haplotype B homozygous wheat varieties. This polymorphic site is named InDel-1478, and the sequence of the 724 bp DNA fragment is shown in SEQ ID No. 2. The polymorphism at this site can be used as a molecular marker to identify the presence of such polymorphic sites in wheat. The specific method is as follows:

[0132] (1) Based on the conserved sequences on both sides of InDel-1478, design specific primer pairs shown in SEQ ID No.3 and SEQ ID No.4.

[0133] (2) Using wheat genomic DNA as a template, PCR amplification was performed using the above-mentioned specific primer pairs; if the amplification product size is 1352 bp, the wheat to be tested is homozygous for haplotype A; if the amplification product size is 628 bp, the wheat to be tested is homozygous for haplotype B; if the amplification product size is 1352 bp and 628 bp, the wheat to be tested is heterozygous for haplotypes A and B, such as Figure 12As shown.

[0134] 3. Using the molecular marker InDel-1478 and its specific primers to perform typing and drought resistance analysis on different wheat varieties.

[0135] Using the drought-resistant wheat variety Yanfu188 as the male parent and the drought-sensitive wheat variety CS (Chinese Spring) as the female parent, a hybrid was formed. The offspring were backcrossed with the female parent for four generations, followed by two generations of self-crossing to obtain near-isogenic lines. BC4F2 generation single plants were subjected to PCR amplification using the method described in step 2 of Example 1 above. If the amplification product size was 1352 bp, the wheat being tested was homozygous for haplotype A; if the amplification product size was 628 bp, the wheat being tested was homozygous for haplotype B; if the amplification product size was both 1352 bp and 628 bp, the wheat being tested was heterozygous for both haplotypes A and B.

[0136] Wheat seeds homozygous for haplotype A and haplotype B from the BC4F2 segregating population were sown in cultivation pots (0.50×0.30×0.2 meters long × wide × deep, containing a cultivation substrate of 3 kg vermiculite and 3 kg peat moss), with 96 plants per pot. They were cultivated in a greenhouse for 21 days under conditions of 16 hours of light and 8 hours of darkness per day, daytime temperature of 14℃ and nighttime temperature of 12℃, and air humidity of 60%. The plants were then subjected to drought treatment (i.e., no watering). One week after the relative soil moisture content (i.e., the volume percentage of water in the soil measured using a soil moisture meter SU-LA(W) purchased from Beijing Mengchuang Weiye Technology Co., Ltd.) reached 0%, the plants were rehydrated. Three days after rehydration, the survival rate of each parent and each genotype in the population was calculated (plants whose above-ground parts failed to turn green after three days of rehydration were defined as dead plants, and those whose above-ground parts turned green after three days of rehydration were defined as surviving plants; the survival rate was the percentage of surviving plants out of the total number of plants). The experiment was repeated three times, and the mean values ​​were used for statistical analysis. The results are shown in Tables 2 and 3. Wheat homozygous haplotype A showed greater drought resistance than wheat homozygous haplotype B. In wheat breeding, wheat homozygous haplotype A with higher drought resistance should be selected for breeding.

[0137] Table 2. Statistical results of parental genotypes and survival rates after drought treatment.

[0138]

[0139] Table 3. Genotypes and survival rates of near-isogenic BC4F2 lines after drought treatment.

[0140]

[0141] The lowercase letters following the results in Tables 2 and 3 represent the results of one-way ANOVA analysis of different genotypes within the population.P A significance level of <0.05 indicates that the difference is not significant if the same lowercase letters are present, and that the difference is significant if the same lowercase letters are absent. The uppercase letters following the results represent the results of one-way ANOVA analysis of different genotypes within the population. P The significance level is <0.01. The presence of identical uppercase letters indicates no significant difference, while the absence of identical uppercase letters indicates a highly significant difference.

[0142] Example 3: Discovery of haplotypes associated with wheat drought resistance

[0143] 1. Drought resistance survey

[0144] A natural variant population of 200 wheat varieties was planted in two cultivation ponds using nutrient soil and vermiculite as the growing medium. Each pond was divided into 200 plots, with 12 seedlings planted in each plot. Watering was stopped when the seedlings had three true leaves, and a drought treatment was implemented until the relative soil moisture content reached zero. Rewatering was then carried out 7 days later, and the survival rate was recorded 3 days after rewatering. The drought phenotypic data used in the statistical analysis were the means of independent replicates. The results are shown in Table 1. Drought resistance was categorized into three types: drought-resistant (survival rate ≥ 40%), drought-sensitive (survival rate < 10%), and intermediate (survival rate ≤ 10% < 40%).

[0145] 2. Statistical analysis of polymorphic sites

[0146] Of the 200 wheat varieties shown in Table 1 TaDi19-1 The 5' untranslated region and promoter region of the gene, a 1.7kb genome fragment, were sequenced in two segments.

[0147] The first segment: SEQ ID No. 1, positions 1-628, using primer sequences SEQ ID No. 19 and SEQ ID No. 20; the second segment: SEQ ID No. 1, positions 393-1329, using primer sequences SEQ ID No. 21 and SEQ ID No. 22. Sequencing results were aligned using MEGA 5.0 (http: / / www.megasoftware.net / ). Nucleotide polymorphism analysis based on the alignment results revealed 17 polymorphic sites, including 9 SNPs and 6 InDels (…). Figure 13 As shown), the minimum allele frequency (MAF) is ≥0.05. Based on the polymorphic site InDel-1478, the 200 wheat varieties can be identified. TaDi19-1 The gene sequence is divided into two haplotypes: haplotype A In-1478 and haplotype B Del-1478.

[0148] Example 4: Activity Verification of Two Monomeric Promoters in Tobacco

[0149] 1. Carrier Construction

[0150] To verify different haplotype sequence pairs TaDi19-1 The influence of gene promoter activity was investigated by cloning the promoter regions (approximately 1.7 kb upstream of the start codon ATG) of haplotypes A and B into the upstream region of the LUC reporter gene in the pGreenII0800-LUC vector, respectively, to construct... pPro-TaDi19-1-HapA ::LUC (monomer-type homozygous) and pPro-TaDi19-1- HapB ::LUC (monomer-type homozygous) plasmid.

[0151] 2. Agrobacterium-mediated transformation

[0152] A transient transformation of tobacco leaves mediated by Agrobacterium was used, and the relative fluorescence intensity of LUC and REN was measured 48 h after transformation. Five biological replicates were set up for the experiment.

[0153] The results are as follows Figure 14 As shown, the relative activity of LUC driven by the promoter of monomeric type B was significantly higher than that of monomeric type A (p<0.01), indicating that monomeric type B has stronger promoter activity. Because TaDi19-1 This gene negatively regulates drought resistance. The result indicates that the promoter activity in haplotype A is low, leading to… TaDi19-1 The expression was downregulated, thereby enhancing the plant's drought resistance.

[0154] Example 5: Expression activity analysis of two haplotype promoters in Arabidopsis thaliana

[0155] 1. Carrier Construction

[0156] Two monomeric promoter sequences (approximately 1.7 kb upstream of the start codon) were cloned into the pCAMBIA1305-GUS vector, replacing the CaMV 35S promoter, to construct... pPro-TaDi19-1-HapA ::GUS (monomeric homozygous) and pPro-TaDi19-1-HapB ::GUS (monomer-isozygous) fusion expression vector.

[0157] 2. Preparation of recipient strains and vector introduction

[0158] Containing recombinant plasmids pPro-TaDi19-1-HapA ::GUS and pPro-TaDi19-1-HapB The plasmid of ::GUS was introduced into Agrobacterium strain GV3101 using the freeze-thaw method.

[0159] Successfully transformed single colonies were cultured in LB medium containing the corresponding antibiotics (Rifampicin 50 mg / L, Kanamycin 50 mg / L), and plasmids were extracted and sequenced to verify that the inserted fragments were correct before being used for Arabidopsis transformation.

[0160] 3. Agrobacterium-mediated inflorescence infection transformation of Arabidopsis thaliana

[0161] Healthy wild-type Arabidopsis thaliana plants (Col-0) were selected and transformed during the early bolting and flowering stage. The successfully transformed Agrobacterium was cultured in LB broth (containing the appropriate antibiotics) until OD... 600 After centrifugation to approximately 0.8, the bacterial cells were collected and resuspended in a transformation solution containing 5% sucrose and 0.05% Silwet L-77. The plants containing inflorescences were inverted and immersed in the bacterial solution for about 30 seconds, then removed and placed in a humid dark environment for 24 hours. Subsequently, normal culture conditions (16 hours light / 8 hours dark) were restored for continued growth to obtain transformed seeds (T0 generation seeds).

[0162] 4. Screening and identification of T0 generation plants

[0163] The harvested T0 generation seeds were stratified at 4°C for 2 days and then sown on 1 / 2 MS medium containing 50 mg / L Hygromycin.

[0164] Seedlings carrying resistance were selected and transplanted into nutrient soil for 3 weeks of cultivation. Leaf DNA was extracted, and PCR amplification was performed to verify whether the GUS fusion gene had integrated into the Arabidopsis genome. PCR-positive plants were identified as transgenic T0 generation positive plants. The primer sequences used were SEQ ID No. 23 and SEQ ID No. 24.

[0165] 5. Obtaining and screening T1 and T2 generation seeds

[0166] Seeds harvested from self-pollination of T0 generation positive plants were designated as T1 generation. T1 generation seeds were cultured under the same resistance selection conditions, and the PCR identification steps described above were repeated to obtain positive individuals. T1 generation positive plants were then self-pollinated to obtain T2 generation seeds. T2 generation positive plants were further self-pollinated to obtain T3 generation seeds. Stable T3 generation transgenic lines were used for subsequent GUS staining experiments.

[0167] The results are as follows Figure 15 As shown, under normal conditions, the GUS signal driven by the haplotype B (HapB) promoter was significantly stronger than that of haplotype A (HapA) in the roots, hypocotyls, and cotyledons, indicating that the haplotype B promoter has higher transcriptional activity. Under treatment with exogenous ABA (100 μM), PEG (20%), and mannitol (200 mM), the GUS staining of haplotype B transgenic plants was significantly enhanced compared to haplotype A, indicating that the haplotype A promoter has lower response activity under stress conditions.

[0168] In summary, the monomeric HapB promoter exhibits high activity. TaDi19-1 Enhanced expression is associated with drought sensitivity; low activity of the haplotype A (HapA) promoter. TaDi19-1 Expression was suppressed, consistent with the drought-resistant phenotype. Furthermore, this result further indicates... TaDi19-1 Genes play a negative regulatory role in drought and the ABA signaling pathway, and differences in their promoter structure directly affect gene expression levels and drought resistance.

[0169] Example 6: McBC-qPCR Analysis of Two Haplotype DNA Sequences

[0170] Analysis using the online website https: / / www.repeatmasker.org / revealed that the 724bp sequence inserted in haplotype A contained Copia Family LTR retroportion. Therefore, to compare different monomer types in TaDi19-1 Differences in DNA methylation levels in gene promoter regions were investigated. From Table 1, 58 drought-resistant materials (monomer type methyl homozygous) and 48 drought-sensitive materials (monomer type methyl homozygous) were selected, and genomic DNA was extracted from leaves at the three-leaf stage.

[0171] The samples were digested using McrBC enzyme, with undigested DNA serving as a control. Quantitative analysis was then performed using qRT-PCR to determine the genetic makeup of wheat. TaActin1 This serves as an internal control. Each sample was tested in triplicate (biological replicates) and triplicate (technical replicates), and the average value was used for statistical analysis.

[0172] The results are as follows Figure 16 As shown, the relative amplification of monomeric DNA A after digestion was significantly lower than that of monomeric DNA B (p<0.01), indicating that the methylation level in this region was higher in monomeric DNA A. Higher methylation modification may reduce promoter activity, thereby inhibiting... TaDi19-1 Transcriptional expression of . In haplotype A TaDi19-1 The expression level was low, consistent with its strong drought resistance phenotype.

[0173] F2 (SEQ ID No. 25): 5'-TGAAGAAGAAATGCAGCCATG-3';

[0174] R2 (SEQ ID No. 26): 5'-GTTAGAAAGTCAACATACTGGCA-3';

[0175] FC (SEQ ID No. 11): 5'-AAATCTGGCATCACACTTTCTAC-3';

[0176] RC (SEQ ID No. 12): 5'-GTCTCAAACATAATCTGGGTCATC-3'.

[0177] Example 7: BSP analysis of two haplotype DNA sequences

[0178] To further verify the methylation level of monomeric A, typical monomeric A material was selected, and genomic DNA was extracted using the CTAB method. After bisulfite treatment, the target fragment was amplified using specific primers targeting the predicted CpG islands approximately 168 bp upstream of the InDel-1478 region. The PCR product was purified and cloned into the pEASY-T1 vector, and 30 positive clones were selected for sequencing analysis. The sequencing results were analyzed for methylation sites and visualized using Kismet software.

[0179] F3 (SEQ ID No. 27): 5'-GGAGTTTAGAAGATAAATTTGAAGAAGAA-3';

[0180] R3 (SEQ ID No. 28): 5'-ATAATAACATATAATTTACCTACCCATATA-3';

[0181] FU (SEQ ID No. 29): 5'-GGAGCCCAGAAGACAAACTTGAAGAAGAA-3';

[0182] RU (SEQ ID No. 30): 5'-ATGATGGCATGTGATTTACCTGCCCATATA-3'.

[0183] The results are as follows Figure 17 As shown, the methylation level at the CG site was 88.86%, at the CHG site it was 49.39%, and at the CHH site it was 3.44%. High levels of CG methylation may be the direct cause of decreased promoter activity in the transposon insertion region, demonstrating that this transposon participates in expression repression by mediating DNA methylation. Combined with McrBC-qPCR results, increased methylation levels of the haplotype methyl promoter lead to decreased gene transcription activity, thereby downregulating expression. TaDi19- 1 The expression of certain substances enhances the plant's drought resistance; while the monomeric ethyl methylation level is low, the promoter activity is high, and the expression level increases, making the plant sensitive to drought stress.

[0184] In summary, differential methylation modification of the InDel-1478 region plays a role in regulating... TaDi19-1 It plays a key role in promoter activity and is an important epigenetic factor causing differences in drought resistance phenotypes among different haplotypes.

[0185] Example 8: TaDi19-1 Gene expression analysis in different haplotype materials

[0186] 1. qRT-PCR detection of parental wheat

[0187] Using drought-resistant parent Yanfu188 (homozygous haplotype A) and drought-sensitive parent CS (homozygous haplotype B) as materials, aboveground parts and roots were sampled at the three-leaf stage. Total RNA was isolated using the TRIZOL (Biotopped) method, followed by DNAseI (Takara) to eliminate genomic contamination. The concentration was then determined using a Nanodrop 1000 (Thermo Scientific product, USA), and 5 μg of each sample was run on 0.8% agarose gel. 1 μg of total RNA was used to synthesize cDNAs using recombinant M-MLV reverse transcriptase with 1 μg Oligo(dT)23 (Promega) as a primer. Specific primers F2 (SEQ ID No. 9) and R2 (SEQ ID No. 10) were used to target the gene. TaDi19-1 qRT-PCR was used to quantify the cDNA of wheat genes. TaActin1 For internal references (SEQ ID No. 11 and SEQ ID No. 12), the results are as follows: Figure 18 As shown, in monomer type B TaDi19-1 The expression level was significantly higher than that of haplotype A (p<0.01), indicating that... TaDi19-1 High expression of this substance is associated with drought sensitivity.

[0188] 2. qRT-PCR detection of wheat offspring from NIL population

[0189] Further analysis of representative plants of different haplotypes in the BC4F2 population. TaDi19-1 Expression level. Sampling time and experimental procedures were the same as in Example 8, and the results were as follows: Figure 19 As shown, the homozygous monotypic plants of type A TaDi19-1 The expression level was significantly lower than that of homozygous haplotype B plants, consistent with their higher drought resistance. This further suggests that the inserted fragment in haplotype A may enhance drought resistance by increasing promoter methylation levels and suppressing gene expression.

[0190] Example 8: Verification of drought resistance phenotypes in parental lines

[0191] Drought-resistant parent Yanfu188 (homozygous haplotype A) and drought-sensitive parent CS (homozygous haplotype B) were transferred to pots containing 250g of nutrient soil. After 21 days of growth under normal conditions, a drought treatment (i.e., watering was stopped) was applied. After 20-30 days, when phenotypic differences became obvious (the leaves of the drought-sensitive parent CS were noticeably withered, while the leaves of the drought-resistant parent Yanfu188 were severely wilted), the plants were rehydrated. Three days after rehydration, the survival rate of each line was recorded (plants that could grow normally and be harvested were defined as surviving plants, and plants that were severely drought-damaged and could not grow normally or be harvested were defined as dead plants; the survival rate was the percentage of surviving plants in each line out of the total number of plants). The experiment was repeated three times, with at least 45 plants in each line in each replicate, and the average value was used for statistical analysis.

[0192] The results are as follows Figure 20 As shown, the survival rate of the drought-resistant parent Yanfu188 (monomer type A homozygous) after rehydration is higher than that of the drought-sensitive parent CS (monomer type B homozygous).

[0193] Three days after rehydration, the survival rates of each plant line were calculated as follows: Figure 21 As shown, the survival rates of the drought-resistant parent Yanfu188 (homozygous haplotype A) and the drought-sensitive parent CS (homozygous haplotype B) were 85.18% and 20.37%, respectively. The survival rate of the drought-resistant parent Yanfu188 (homozygous haplotype A) was significantly higher than that of the drought-sensitive parent CS (homozygous haplotype B).

[0194] Example 9: Validation of drought resistance phenotype in NIL population

[0195] NILIn-1478 (homozygous monomeric type A) and NILDel-1478 (homozygous monomeric type B) were transferred to pots containing 250g of nutrient soil. After 21 days of growth under normal conditions, a drought treatment (i.e., watering was stopped) was applied. After 20-30 days, the phenotypic differences became obvious, i.e., NILIn-1478 showed significant differences. Del-1478 (Monotype homozygous) Leaves show obvious signs of drying out, while the drought-resistant parent NIL In-1478 (Homozygous monotypic type A) When leaves were severely wilted, the plants were rehydrated. Three days after rehydration, the survival rate of each line was counted (plants that could grow normally and be harvested were defined as surviving plants, and plants that were severely affected by drought and could not grow normally or be harvested were defined as dead plants; the survival rate was the percentage of surviving plants in each line out of the total number of plants). The experiment was repeated three times, with at least 45 plants in each line in each replicate, and the average value was used for statistical analysis.

[0196] The results are as follows Figure 22 As shown, it can be seen that NIL In-1478 The survival rate after rehydration is higher than that of NIL. del-1478Three days after rehydration, the survival rate of each plant line was calculated as follows: Figure 23 As shown, it can be seen that NIL In-1478 and NIL Del-1478 The survival rates were 81.4% and 27.7%, respectively, for NIL. In-1478 The survival rate was significantly higher than that of NIL. Del-1478 .

[0197] The above results indicate that after the insertion of a 724 bp fragment into the promoter region by the monomeric A, the methylation level increases, promoter activity decreases, and transcriptional activity decreases, leading to... TaDi19-1 Expression decreases, thereby enhancing wheat drought resistance; while the monomeric B promoter has higher activity. TaDi19-1 Increased expression levels reduce the plant's drought resistance.

[0198] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a reagent for detecting InDel molecular markers related to drought resistance in wheat in the identification or auxiliary identification of wheat drought resistance, characterized in that, Using the genomic DNA of wheat as a template, InDel molecular markers related to wheat drought resistance were amplified. When the amplified product contained the sequence shown in SEQ ID No. 2, the drought resistance was better than that of the sequence not shown in SEQ ID No.

2. The InDel molecular marker is defined as the insertion or deletion of a nucleotide sequence such as the fragment shown in SEQ ID No. 2 between 245-246 bp of the sequence shown in SEQ ID No.

1.

2. A method for identifying or assisting in the identification of wheat genotypes, characterized in that, The method includes using the genomic DNA of wheat as a template to amplify InDel molecular markers related to wheat drought resistance. When the amplification product contains the sequence shown in SEQ ID No. 2, it is determined to be drought-resistant monomer type A, and when the sequence shown in SEQ ID No. 2 is missing, it is determined to be non-drought-resistant monomer type B. The InDel molecular marker is defined as the insertion or deletion of a nucleotide sequence such as the fragment shown in SEQ ID No. 2 between 245-246 bp of the sequence shown in SEQ ID No.

1.

3. The method according to claim 2, characterized in that, The primer pair used for amplification includes an upstream primer with nucleotide sequences as shown in SEQ ID No. 3 and a downstream primer with SEQ ID No.

4.

4. The method according to claim 3, characterized in that, The amplification product of the drought-resistant monomer type A is 1352bp; the amplification product of the non-drought-resistant monomer type B is 628bp.

5. The method according to claim 2, characterized in that, The amplification program includes: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 1 min, 38 cycles; and 72℃ extension for 5 min.

6. The application of a reagent for detecting InDel molecular markers related to drought resistance in wheat in improving wheat drought resistance, characterized in that, Using the genomic DNA of the wheat to be tested as a template, InDel molecular markers related to drought resistance in wheat were amplified. When the amplification product contained the sequence shown in SEQ ID No. 2, the drought resistance was better than that of wheat without the sequence shown in SEQ ID No.

2. Wheat of drought-resistant haplotype A containing the sequence shown in SEQ ID No. 2 in the amplification product was selected for breeding. The InDel molecular marker is defined as the insertion or deletion of a nucleotide sequence fragment as shown in SEQ ID No. 2 between 245-246 bp of the sequence shown in SEQ ID No.

1.

7. The application of a reagent for detecting nDel molecular markers related to drought resistance in wheat in the breeding or assisted breeding of drought-resistant wheat varieties, characterized in that, Using the genomic DNA of wheat as a template, InDel molecular markers related to wheat drought resistance were amplified. When the amplified product contained the sequence shown in SEQ ID No. 2, the drought resistance was better than that of the sequence not shown in SEQ ID No.

2. The InDel molecular marker is defined as the insertion or deletion of a nucleotide sequence such as the fragment shown in SEQ ID No. 2 between 245-246 bp of the sequence shown in SEQ ID No.

1.

8. A method for screening drought-resistant wheat varieties, characterized in that, This includes using the genomic DNA of the wheat to be tested as a template to amplify the InDel molecular markers related to wheat drought resistance, selecting drought-resistant monomer type A containing the sequence shown in SEQ ID No. 2 in the amplification product, and discarding non-drought-resistant monomer type B that lacks the sequence shown in SEQ ID No. 2 in the amplification product; The InDel molecular marker is defined as the presence of an insertion or deletion of a nucleotide sequence, such as the fragment shown in SEQ ID No. 2, between 245-246 bp of the sequence shown in SEQ ID No. 1.

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