Wheat powdery mildew related gene TaWIR1b and application thereof

Abstract

This invention belongs to the field of bioengineering technology and relates to a gene related to wheat powdery mildew. TaWIR1b And its applications. Genes TaWIR1b The sequence is shown in SEQ ID NO:1, in gene form. TaWIR1b Based on a partial sequence SEQ ID NO:6, the 245th base at the 5' end, starting from the first ATG, is either G or A. Type A causes an intron-preserving alternative splice, resulting in premature termination of amino acid translation. This invention provides a wheat powdery mildew gene. TaWIR1b The sequence, the protein sequence encoded by the gene, and identification. TaWIR1b Gene-specific primer pairs were used, and dCAPS markers were developed for identification. TaWIR1b TaWIR1b Different allele types; this invention also constructed a VIGS silencing vector based on the above genes for gene function verification. The discovery, identification, and application of wheat powdery mildew-related genes in this invention not only help to reveal the genetic basis of wheat powdery mildew, but also play a very important role in improving wheat resistance using genetic engineering technology.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a wheat powdery mildew-related gene TaWIR1b and its application. Background Technology

[0002] Bread wheat (Triticum aestivum L., 2n=6x=42, AABBDD) is one of the major crops and plays a vital role in global food security (Lobell DB, Gourdji SM. The influence of climate change on global crop productivity. Plant Physiol. 2012 Dec;160(4):1686-97. doi:10.1104 / pp.112.208298.). Powdery mildew of wheat, caused by the grass blumeria *Blumeria graminis* f.sp. *tritici* (Bgt), is a global fungal disease. Infection with powdery mildew leads to leaf death and reduces tiller number, ear setting rate, number of grains per ear, and thousand-grain weight, severely threatening wheat yield and quality (Feng W., Li X., Liu W., Wang X., Wang C., Gou T. Effects of powdery mildew infection on grainquality traits and yield of winter wheat. J. Triticeae Crops. 2014; 34:1706–1712.; Singh RP, Singh PK, Rutkoski J, Hodson DP, He X, LN, MS, Huerta-Espino J. Disease Impact on Wheat Yield Potential and Prospects of Genetic Control. Annu Rev Phytopathol. 2016 Aug 4; 54:303-22. Currently, the control of wheat powdery mildew in production still mainly relies on chemical spraying and reasonable planting density. However, the use of chemical agents such as triadimefon and triadimefon not only increases field input costs, but also the problems of pesticide residues and environmental pollution caused by excessive application of pesticides cannot be ignored. Especially when powdery mildew breaks out in large numbers, chemical agents are difficult to achieve ideal control effects, and repeated excessive use of pesticides can also lead to drug resistance (Jankovics T, Komáromi J, Fábián A, K, Vida G, Kiss L. New Insights into the Life Cycle of the Wheat Powdery Mildew: Direct Observation of Ascosporic Infection in Blumeria graminis f.sp.tritici. Phytopathology. 2015 Jun; 105(6):797-804.; Zheng Hongyuan. Functional Study on Regulation of Wheat Powdery Mildew Resistance by TuMYB46L-TuACO3 Module [D]. Henan Agricultural University, 2019.). Relatively speaking, breeding powdery mildew-resistant varieties of wheat is currently the most economical, effective, and environmentally friendly method of control (Selter, L., Shatalina, M., Singlea, J., Keller, B. (2014). Identification and Implementation of Resistance: Genomics-Assisted use of Genetic Resources for Breeding Against Powdery Mildew and Stagonospora Noodorum Blotch in Wheat. In: Tuberosa, R., Graner, A., Frison, E. (eds) Genomics of Plant Genetic Resources. Springer, Dordrecht. https: / / doi.org / 10.1007 / 978-94-007-7575-6_15).

[0003] To date, 68 powdery mildew resistance genes (Pm1-Pm68) have been located and formally named (McIntoshRA, Dubcovsky J, Rogers WJ, Morris C, Xia XC. 2017. Catalogue of gene symbols for wheat: 2017 supplement (KOMUGI Wheat Genetic Resource Database). [WWW document] URL https: / / shigen.nig.ac.jp / wheat / komugi / genes / ;Zhang R,Xiong C,Mu H,etal. Pm67, a new powdery mildew resistance gene transferred from Dasypyrum villosum chromosome 1V to common wheat (Triticum aestivum L.)[J]. The Crop Journal, 2021,9(4):882-888.;He,H.,Liu,R.,Ma,P.etal. Characterization of Pm68, a new powdery mildew resistance gene on chromosome 2BS of Greek durum wheat TRI1796. Theor Appl Genet 134,53–62 (2021). However, only 12 genes have been cloned (Si Guan, Zhao Zhiyong, Bao Haizhu, et al. Current status of wheat germplasm resources resistant to powdery mildew and research progress on resistance genes [J]. Ningxia Agricultural and Forestry Science and Technology, 2022, 63(06):14-20.), and most of them are nucleic acid-binding, leucine-rich repeat (NLR) encoded resistance (R) genes.R genes often function as specialized resistance genes targeting specific pathogens (Dangl JL, Horvath DM, Staskawicz BJ. Pivoting the plant immune system from dissection to deployment. Science. 2013 Aug 16; 341(6147):746-51.), directly or indirectly recognizing pathogenic effectors and triggering plant immunity (Dangl JL, Jones JD. Plant pathogens and integrated defense responses to infection. Nature. 2001 Jun 14; 411(6839):826-33.). However, due to the rapid mutation and evolution of powdery mildew, the function of specialized resistance genes is lost quickly. Once widely adopted, their resistance is quickly destroyed by new mutant strains. Currently, among the wheat varieties promoted in my country, the effective or locally effective powdery mildew resistance genes are mainly a few major genes such as Pm2, Pm4, Pm21, and Pm52. Furthermore, some alleles of Pm2 and Pm4 have gradually lost their resistance in some areas. Therefore, manipulating and disrupting the susceptibility (S) gene to mediate plant immunity is currently a more attractive option (Oliva R, Ji C, Atienza-Grande G, Huguet-Tapia JC, Perez-Quintero A, Li T, Eom JS, Li C, Nguyen H, Liu B, Auguy F, Sciallano C, Luu VT, DossaGS, Cunnac S, Schmidt SM, Slamet-Loedin IH, Vera Cruz C, Szurek B, Frommer WB, WhiteFF, Yang B. Broad-spectrum resistance to bacterial blight in rice using genome editing. Nat Biotechnol. 2019 Nov; 37(11):1344-1350.).In 2014, Chinese scientists, for the first time, used genome editing technology to simultaneously mutate three Mlo homologous genes in wheat, obtaining wheat materials with extremely significant broad-spectrum resistance to powdery mildew (Wang, Y., Cheng, X., Shan, Q. et al. Simultaneous editing of threehomoeoalleles in hexaploid bread wheat confers heritable resistance topowdery mildew. Nat Biotechnol 32, 947–951 (2014).). In 2022, the Gao Caixia / Qiu Jinlong team created the Tamlo-R32 mutant using dual "gene scissors," solving the negative impact of conventional Mlo mutants on wheat growth and obtaining new materials that are both highly resistant to powdery mildew and high-yielding (Li, S., Lin, D., Zhang, Y. et al. Genome-edited powdery mildew resistance in wheat without growth penalties. Nature 602, 455–460 (2022).).

[0004] Therefore, using genome editing technology to precisely manipulate susceptible genes and create broad-spectrum disease-resistant and high-yielding germplasm resources, thus enabling more convenient, faster, and ultra-precise crop breeding and improvement programs, is a more attractive and meaningful option at this stage. Summary of the Invention

[0005] The purpose of this invention is to provide a wheat powdery mildew-related gene TaWIR1b, its expression product, silencing vector, etc., and to apply it to wheat powdery mildew resistance breeding, in order to provide more options for breeding wheat varieties with multiple disease resistance genes.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] This invention has identified and obtained a wheat powdery mildew-related gene, TaWIR1b, whose nucleotide sequence in the wheat genome is shown in SEQ ID NO:1, or its full-length CDS nucleotide sequence is as follows:

[0008] a: A nucleotide sequence as shown in SEQ ID NO:2; or

[0009] b: A nucleotide sequence with equivalent function derived from the nucleotide sequence shown in SEQ ID NO:2.

[0010] This includes DNA molecules that hybridize with the DNA sequence of SEQ ID NO:2 under stringent conditions and encode proteins that control wheat powdery mildew-related traits; or DNA molecules that have more than 90% homology with the DNA sequence of SEQ ID NO:2 and encode proteins that control wheat powdery mildew-related traits.

[0011] This invention provides a protein encoded by the wheat powdery mildew-related gene TaWIR1b, the amino acid sequence of which is as follows:

[0012] (1) The amino acid sequence as shown in SEQ ID NO:3; or

[0013] (2) A sequence of an active fragment or a conserved variant obtained by adding, deleting or replacing one or more amino acids based on the amino acid sequence shown in SEQ ID NO:3.

[0014] This invention also provides a product for identifying the above-mentioned wheat powdery mildew-related gene TaWIR1b, said product comprising:

[0015] The nucleotide sequence is as shown in the upstream primer of SEQ ID NO:4, and

[0016] Downstream primers with nucleotide sequences as shown in SEQ ID NO:5. And reagents or kits for said primers.

[0017] This invention further provides a method for detecting wheat powdery mildew alleles using the above-mentioned specific primers, comprising the following steps:

[0018] Step 1: Extract genomic DNA from the wheat sample using the CTAB method;

[0019] Step 2: Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using the upstream primer shown in SEQ ID NO:4 and the downstream primer shown in SEQ ID NO:5, to obtain the PCR amplification product shown in SEQ ID NO:6. Then, the following judgment was made:

[0020] Using the nucleotide sequence of the PCR product as shown in SEQ ID NO:6 as a standard, the nucleotide at position 245, starting from the first ATG at the 5' end, is determined to be either G or A to identify the protein encoded by the wheat being tested. A base change from G to A, i.e., a base mutation at the junction of the intron and exon start positions, causes the intron in the nucleotide sequence of SEQ ID NO:1 to be unable to be spliced, resulting in an intron-preserving type of alternative splicing mutation. The preserved translation of the intron ultimately leads to premature termination of the TGA encoding at positions 178-180, starting from the ATG at the 5' end, 2000 bp after the intron in the nucleotide sequence of SEQ ID NO:1. The corresponding allele genotype is named TaWIR1b.1. The nucleotide sequence corresponding to the G genotype is spliced ​​and translated normally, and its corresponding allele genotype is named TaWIR1b.2.

[0021] Extensive phenotypic identification results show that plants with the TaWIR1b.1 allele have good resistance to wheat powdery mildew, while plants with the TaWIR1b.2 allele exhibit susceptibility to wheat powdery mildew. Therefore, the discovery of the functions of different alleles of the TaWIR1b.1 gene will play a very important role in identifying the powdery mildew resistance of different wheat varieties using genetic engineering technology, and will also contribute to more in-depth research on the genetics and molecular biology of wheat disease resistance breeding.

[0022] This invention provides a composition for identifying TaWIR1b allelic differential loci, the composition comprising:

[0023] The upstream primer with the nucleotide sequence shown in SEQ ID NO:4, and the downstream primer with the nucleotide sequence shown in SEQ ID NO:5, and the restriction endonuclease EcoRII used. And perform the judgment as described in (I) or (II) below:

[0024] (I) The wheat germplasm resources (species / lines) whose amplified nucleotide fragments, after being digested with EcoRII, still have a length of 408 bp, are of the TaWIR1b.1 allele type. Statistically, their resistance to wheat powdery mildew is significantly higher than that of wheat germplasm resources (species / lines) whose fragments remain at 263 bp after digestion.

[0025] (II) The wheat germplasm resources (species / lines) with a nucleotide fragment of 263 bp after EcoRII digestion have the TaWIR1b.2 allele type. Statistically, their resistance to wheat powdery mildew is significantly lower than that of wheat germplasm resources (species / lines) with a fragment of 408 bp after digestion.

[0026] The aforementioned wheat powdery mildew-related gene TaWIR1b, TaWIR1b protein, products for identifying the aforementioned wheat powdery mildew-related gene TaWIR1b, or compositions for identifying TaWIR1b allelic differential loci can be applied to:

[0027] (1) To identify or assist in the identification of wheat resistance to powdery mildew;

[0028] (2) Disease-resistant wheat breeding;

[0029] (3) Construction of wheat genetic map;

[0030] (4) Analysis of genetic diversity of wheat germplasm resources;

[0031] (5) Identification of wheat germplasm resources.

[0032] This invention designs a virus-induced gene silencing recombination (such as BSMV-VIGS) vector constructed from the aforementioned gene.

[0033] The primer sequences for amplifying the silenced fragment are:

[0034] The recombinant silencing vector is obtained by inserting the nucleotide sequence shown in SEQ ID NO:9 between the PacI and NotI restriction sites of the γ vector (the γ part of the BSMV viral vector) into the upstream primer with the nucleotide sequence shown in SEQ ID NO:7 and the downstream primer with the nucleotide sequence shown in SEQ ID NO:8.

[0035] Further, the viral vector and recombinant plasmid were linearized and transcribed in vitro to obtain RNA viruses with different components. Then, the different components of RNA viruses were prepared into infection premixes, and corresponding treatment groups and control groups were set up to inoculate wheat seedling leaves to silence the TaWIR1b gene in order to verify the function of this gene.

[0036] After silencing the TaWIR1b gene in wheat plants using a TaWIR1b gene silencing recombinant vector, the following assessments were made regarding the phenotype of the silenced plants:

[0037] Compared with WT plants, the resistance to wheat powdery mildew in silent plants was significantly enhanced, indicating that the TaWIR1b gene plays a negative regulatory role in the material. Such materials are mostly susceptible to wheat powdery mildew.

[0038] This invention also provides the application of the wheat powdery mildew gene TaWIR1b in wheat disease resistance breeding.

[0039] Compared with the prior art, the main beneficial technical effects of the present invention are as follows:

[0040] 1. This invention discloses and confirms for the first time a new wheat powdery mildew-related gene, named TaWIR1b, which is located on the short arm of wheat chromosome 5D. The protein expressed by this gene can regulate wheat resistance to powdery mildew.

[0041] 2. This invention clarifies the DNA sequence, CDS sequence, and encoded protein sequence of the wheat powdery mildew-related gene TaWIR1b, laying a technical foundation for the application of wheat disease resistance breeding.

[0042] 3. The specific primers and restriction endonucleases used in this invention can be directly used to detect the TaWIR1b allele genotype of wheat powdery mildew-related genes, which is beneficial for screening superior resistance materials for wheat powdery mildew; the breeding of wheat disease-resistant varieties and the creation of superior germplasm resources can achieve early screening at the molecular level, saving time and costs.

[0043] 4. This invention utilizes VIGS technology to silence the wheat powdery mildew-related gene TaWIR1b for the first time, verifying the role of the TaWIR1b gene in wheat powdery mildew resistance breeding.

[0044] 5. This invention helps to reveal the molecular genetic basis of wheat powdery mildew, and plays an important role in using genetic engineering technology to detect and improve wheat varieties, providing a new approach for breeding disease-resistant and safe new wheat varieties. Attached Figure Description

[0045] Figure 1 These are Manhattan and Q-Q plots from the genome-wide association analysis of wheat powdery mildew in this invention. A: GWAS Manhattan plot; B: GWAS Q-Q plot; C: GWAS haplotype analysis plot. Note: Chr.5D refers to wheat chromosome 5D.

[0046] Figure 2 This is the QTL mapping map for wheat powdery mildew according to the present invention. Note: Chr.5D refers to wheat chromosome 5D.

[0047] Figure 3 A bar chart showing the basal expression levels of differentially expressed genes in leaves under different inoculation treatments within the localized region of the resistant mixed pool.

[0048] Figure 4This image shows a bar graph of the relative expression levels of three candidate genes in a VIGS silencing assay (recipient material was the susceptible parent, *Chinese Spring*) and photographs of their resistance responses to wheat powdery mildew. The left image shows the VIGS silencing qRT-PCR, and the right image shows the wheat powdery mildew resistance phenotype after silencing. BSMV:γ0 represents control plants inoculated with empty BSMV virus vectors; BSMV:TaWIR1a / TaWIR1b / TaWIR1c represents silenced plants inoculated with recombinant vectors BMSV-TaWIR1a, BMSV-TaWIR1b, and BMSV-TaWIR1c; and WT represents control plants not inoculated with BSMV virus.

[0049] Figure 5 The expression patterns of the TaWIR1b gene at different time points in the E09 parental strain *Chinese Spring* after inoculation.

[0050] Figure 6 Electrophoresis results of dCAPS-labeled enzyme digestion developed based on TaWIR1b allele types. 1-4: susceptible lines; 5-8: resistant lines; 9: negative control (water); 10: Marker D2000; 11: Marker 50bp.

[0051] Figure 7 This is a graph showing the haplotype analysis results of the TaWIR1b gene. R indicates resistance, and S indicates susceptibility.

[0052] Figure 8 This is a bar chart showing the relative expression levels of TaWIR1b in VIGS silencing experiments on different recipient materials (Zhou18, XN18, WM208). In the figure, Zhou18 represents Zhoumai 18; XN18 represents Xinong 18; and WM208 represents Weimin 208. γ0-Zhou18, γ0-XN18, and γ0-WM208 represent plants inoculated with empty BSMV virus using Zhoumai 18, Xinong 18, and Weimin 208 as recipient materials, respectively. TaWIR1b-Zhou18, TaWIR1b-XN18, and TaWIR1b-WM208 represent silenced plants inoculated with recombinant vector BMSV-TaWIR1b virus using Zhoumai 18, Xinong 18, and Weimin 208 as recipient materials, respectively.

[0053] Figure 9is a comparative photograph of the resistance of different recipient materials (Zhou18, XN18, WM208) to wheat powdery mildew in the VIGS silencing test of TaWIR1b. On the left of the figure: Zhou18: Zhoumai 18; XN18: Xinong 18; WM208: Weimin 208. BSMV:γ0 represents plants inoculated with empty BSMV virus, BSMV:TaWIR1b represents silenced plants inoculated with recombinant vector BSMV-TaWIR1b virus. On the right of the figure: γ0: Zhou18, γ0: XN18; γ0: WM208 represent plants inoculated with empty BSMV virus using Zhoumai 18, Xinong 18, and Weimin 208 as recipient materials respectively. TaWIR1b: Zhou18, TaWIR1b: XN18, TaWIR1b: WM208 respectively represent silenced plants inoculated with recombinant vector BSMV-TaWIR1b virus using Zhoumai 18, Xinong 18, and Weimin 208 as recipient materials. Detailed Description of the Embodiments

[0054] The present invention is described in more detail below through specific embodiments to facilitate understanding of the technical solutions of the present invention, which is not intended to limit the protection scope of the present invention.

[0055] Unless otherwise specified, all instruments and equipment involved in the following examples are conventional instruments and equipment; all involved reagents are commercially available conventional reagents; all involved test methods are conventional methods unless otherwise specified.

[0056] The phenotypic identification tests in the following examples are all set up with three repeated experiments, and the results are averaged. Primer synthesis and sequencing were all completed by Sangon Biotech (Shanghai) Co., Ltd.

[0057] The wheat materials involved below are:

[0058] A natural population consisting of 163 wheat varieties (lines) from the Huanghuai wheat region (Table 1) was genotyped using a genome-wide 90K SNP chip (Beijing Compass Biotechnology Co., Ltd.), and this population was used for genome-wide association study (GWAS).

[0059] Table 1: List of 163 wheat materials from the Huanghuai wheat region

[0060]

[0061]

[0062] The F population consisting of 237 families generated by crossing Chinese Spring and L19 2：3 (referred to as CL population for short) is used for QTL mapping.

[0063] Chinese Spring: A susceptible parent of powdery mildew, used in VIGS assays to screen candidate genes and verify the effect of the TaWIR1b gene.

[0064] Zhoumai 18, XN18, and WM208: These are genotypes and phenotypes of highly susceptible wheat powdery mildew materials, used in the VIGS experiment to verify the effect of the TaWIR1b gene.

[0065] Jingshuang 16: A material highly susceptible to powdery mildew in both wheat seedling and mature stages, used as a control for indoor propagation of powdery mildew and indoor identification experiments.

[0066] Example 1: Identification of powdery mildew resistance in different wheat varieties

[0067] 1. Selection of test strains and preparation of experimental soil

[0068] The strain used in the experiment was Powdery mildew E09 (gifted by Li Hongjie's team from the Institute of Crop Science, Chinese Academy of Agricultural Sciences). It is a highly virulent physiological race of powdery mildew currently prevalent in North China, characterized by its wide range of infection and strong pathogenicity.

[0069] The soil used in the experiment was indoor horticultural grade nutrient soil: vermiculite: perlite = 30:1:1. The soil was sterilized in an autoclave at 121℃ and 0.1Mpa for 1 hour before use.

[0070] 2. Material planting and resistance identification

[0071] Pathogen propagation: Propagation was carried out using indoor susceptible host parasitism. First, the susceptible control Jingshuang 16 was cultured in a light incubator until the one-leaf-one-heart stage. Then, wheat leaves carrying the E09 strain of Brycetes powdery mildew were rubbed onto the leaves of the susceptible control Jingshuang 16. Finally, the pathogens were cultured and propagated in a closed greenhouse free of other pathogens.

[0072] Material planting and cultivation: Place 180g of sterile soil in an 8cm*8cm square culture box, with the groove facing down. Arrange 12 wheat grains evenly in a 3*4 pattern, cover with 30g of soil, and repeat each treatment three times. Place the boxes in trays, ensuring at least one susceptible control (Jingshuang 16) per tray. Thoroughly water the bottom of each tray and incubate under alternating light and dark conditions (16h / 8h), at 25℃ / 15℃ and 60-80% humidity. Three days after emergence, observe the growth of the seedlings, remove weak seedlings, and inoculate with powdery mildew fungus when the seedlings reach the one-leaf-one-heart stage.

[0073] Material investigation and evaluation: The leaf phenotype of the plants was investigated 7-10 days after inoculation. The plants were identified according to the IT level of 0-4, where IT=0 (immune); IT=0 (near-immune); IT=1 (highly resistant); IT=2 (moderately resistant); IT=3 (moderately susceptible); and IT=4 (highly susceptible).

[0074] Example 2: Localization of the wheat powdery mildew resistance locus QPM.CL

[0075] Phenotypic identification at the seedling stage differentiated the different response types of 163 wheat varieties to wheat powdery mildew. Further analysis, combined with population 90K SNP chip information (Beijing Compson Biotechnology Co., Ltd.), followed by quality control, and genome-wide association analysis (GWAS) of wheat powdery mildew resistance, identified SNP loci significantly associated with wheat powdery mildew on wheat chromosome 5D. Figure 1 Further utilizing the aforementioned SNP sites, a BLAST analysis was performed on the Chinese spring reference genome information from the wheat genome database (http: / / 202.194.139.32 / ). It was found that the locked regions of these SNP sites are located between 44,698,893 bp and 49,019,751 bp (≈4.3 Mb) on wheat chromosome 5D.

[0076] Simultaneously, the seedling wheat powdery mildew resistance of the CL population and its parents was identified. Statistical analysis showed that the segregation of resistance in the population conformed to the segregation ratio controlled by a single gene (F2 = 3:1; F2 = 3:1). 2:3 =1:2:1), as shown in Table 2. Then, according to CLF... 2:3 Phenotypic data from the population greenhouse were used to select extremely resistant and susceptible families to construct resistant-susceptible pools. The wheat 660K SNP array (Sun C, Dong Z, Zhao L, Ren Y, Zhang N, Chen F. The Wheat 660K SNP array demonstrates great potential for marker-assisted selection in polyploid wheat. Plant Biotechnol J. 2020 Jun; 18(6):1354-1360.) was used to detect the parental and resistant-susceptible mixed pools. BSA-Seq analysis of the parental and mixed pools showed that differentially expressed SNPs were mainly concentrated on wheat chromosome 5D. Furthermore, 10 KASP markers were developed on chromosome 5D (Table 3) for CLF. 2:3 The population was tested, and combined with the phenotypic data of the population, a major QTL affecting powdery mildew resistance was located on chromosome 5D of the population using the complete interval mapping method, which is between markers cfd81-A010610 (Table 4). Figure 2 This corresponds to the range of approximately 10.5Mb from 44,567,002bp to 55,049,899bp in the Chinese Spring v1.0 database (http: / / 202.194.139.32 / ).

[0077] The results of QTL mapping and GWAS analysis showed that there is a major QTL for powdery mildew resistance in the interval of 44.70Mb to 49.02Mb (approximately 4.32Mb) on the wheat 5DS chromosome, named QPM.CL.

[0078] Table 2: Genetic identification of powdery mildew resistance in Chinese Spring, L19 and their offspring populations

[0079]

[0080]

[0081] Table 3: KASP Tag Development

[0082]

[0083] Table 4: QTL of the CL population

[0084]

[0085] Example 3: Localization and Cloning of the Wheat Powdery Mildew-Related Gene TaWIR1b

[0086] Referring to the Chinese Spring Reference Genome v1.0 database (http: / / 202.194.139.32 / ), there are 139 annotated genes in the co-localized region of QPM.CL. Combined with the transcriptome results of the mixed pool of resistant and susceptible genes, 13 differentially expressed genes were screened within the co-localized region. Simultaneously, using the Chinese Spring RNA-Seq database (http: / / 202.194.139.32 / ), leaf RNA-Seq data for these 13 differentially expressed genes were retrieved. Using a leaf expression level Transcript per million (TPM) value >3 and differential expression in at least two control treatments as the screening criteria, three candidate genes were further screened: TraesCS5D02G049200 (named TaWIR1a), TraesCS5D02G050100 (named TaWIR1c), and TraesCS5D02G050200 (named TaWIR1b), and further functional validation was performed. Figure 3 ).

[0087] The susceptible parent plant, *Chinese Spring*, was selected as the recipient material. The wheat genes TaWIR1a, TaWIR1b, and TaWIR1c were silenced using VIGS technology (see Example 5 for expression vector construction and experimental procedures), and the plants were then inoculated with *Brucella powdery mildew* E09. The results showed that compared to the control plants WT (uninoculated control plants) and BSMV-γ0 (inoculated plants with empty BSMV virus), the powdery mildew phenotype of TaWIR1a and TaWIR1c-silenced plants did not change significantly, while the degree of powdery mildew infection in TaWIR1b-silenced plants was significantly reduced. Figure 4 The VIGS transient silencing experiment results indicate that the TaWIR1b gene negatively regulates wheat powdery mildew resistance.

[0088] Furthermore, after inoculation with *Brucea javanica* E09 at different time points, qRT-PCR results showed that TaWIR1b gene expression in the susceptible parent *Prunus chinensis* was induced to be upregulated in *Prunus javanica* 24 h after inoculation with *Brucea javanica* E09. Figure 5 Therefore, TaWIR1b is considered to be a gene associated with wheat powdery mildew.

[0089] Referring to the NCBI (https: / / www.ncbi.nlm.nih.gov / ) and EnsemblPlants (https: / / plants.ensembl.org) databases for the genome of Chinese spring plants, primers were designed using Primer5 software to amplify a portion of the gene sequence. The forward primer of the PCR amplification primer pair was the upstream primer with the nucleotide sequence shown in SEQ ID NO:4, such as: 5'-GTACCAGATTACGCTCATATGGCGTCCCACAGTGCT-3', and the reverse primer was the downstream primer with the nucleotide sequence shown in SEQ ID NO:5, such as: 5'-CAGCTCGAGCTCGATGGATCCTTAGGGCTGGCTACCCGC-3'.

[0090] The genomic DNA of wheat *Cyperus chinensis* was amplified using primers shown in SEQ ID NO:4 and SEQ ID NO:5. The results showed that the obtained sequence was identical to the corresponding *Cyperus chinensis* sequence in the EnsemblPlants (https: / / plants.ensembl.org) database. The PCR amplification system is shown in Table 5, and the PCR amplification procedure is shown in Table 6.

[0091] Table 5: PCR amplification system of primer pairs for partial TaWIR1b gene sequence

[0092] 2×phanta buffer 12.5μL dNTP 0.5μL Phanta Enzyme 0.5μL Primer F 0.5μL Primer R 0.5μL DNA 1μL <![CDATA[ddH2O]]> To 25μL

[0093] Table 6: PCR amplification program for partial TaWIR1b gene sequence using primer pairs

[0094] 95℃ 5min 1 95℃ 30s 58℃ 30s 37 72℃ 15s 72℃ 5min 1 12℃ Insulation

[0095] Sequencing verification revealed that the partial sequence of the TaWIR1b gene obtained by primers shown in SEQ ID NO:4 and SEQ ID NO:5—SEQ ID NO:6—exhibited polymorphism between resistant and susceptible materials. In the resistant materials, the 245th base from the first ATG at the 5' end (i.e., the 263rd base of SEQ ID NO:6) was mutated from G to A, forming an alternative splicing mutation. The powdery mildew phenotypes of the resistant and susceptible materials showed significant differences. Therefore, the full-length genomic DNA containing SEQ ID NO:6 (the gene shown in SEQ ID NO:1) is a gene associated with the wheat powdery mildew trait.

[0096] Example 4: Development and Application of TaWIR1b Allele Markers

[0097] Based on the G / A separation of different materials from the 245th base starting from the first ATG at the 5' end of the sequence shown in SEQ ID NO:6, a dCAPS marker was developed to detect this mutation site. Specific primer pairs for amplifying the mutation site include:

[0098] The upstream primer sequence 5'-GTACCAGATTACGCTCATATGGCGTCCCACAGTGCT-3' with nucleotide sequences as shown in SEQ ID NO:4 and the downstream primer sequence 5'-CAGCTCGAGCTCGATGGATCCTTAGGGCTGGCTACCCGC-3' with nucleotide sequences as shown in SEQ ID NO:5.

[0099] The amplified products were digested using the specific restriction endonuclease EcoRII. Wheat varieties / lines with a differentially expressed sequence at site A remaining at 408 bp after EcoRII digestion were named the TaWIR1b.1 allele, and wheat varieties / lines with a differentially expressed sequence at site G remaining at 263 bp after EcoRII digestion were named the TaWIR1b.2 allele (see...). Figure 6 The disease resistance of the plants corresponding to the two genotypes differed significantly.

[0100] The developed TaWIR1b gene dCAPS marker primer pair (primer pairs shown in SEQ ID NO:4 and SEQ ID NO:5) was further tested in CLF. 2:3The results of the detection and verification in the population are shown in Table 7. The TaWIR1b.1 and TaWIR1b.2 alleles showed clear segregation in the population, exhibiting a segregation ratio of approximately 3:1 controlled by a single gene. The corresponding differences in wheat powdery mildew resistance were significant. This marker can serve as a detection marker for the TaWIR1b gene in wheat powdery mildew and as an auxiliary selection marker for plant resistance.

[0101] Table 7: TaWIR1b allele detection and phenotypic differences in the CL population

[0102] L19 TaWIR1b.1 1 0 Chinese Spring TaWIR1b.2 1 4 Disease-resistant system TaWIR1b.1 173 1 Disease System TaWIR1b.2 61 4

[0103] Simultaneously, the TaWIR1b gene was sequenced in 480 varieties / lines (Table 8), obtaining polymorphic information for 357 TaWIR1b genes. Haplotype analysis revealed that 14 materials were of the HapA allele, i.e., TaWIR1b.1. Seedling resistance identification showed that the HapA allele, i.e., TaWIR1b.1, exhibited good resistance to wheat powdery mildew. The HapB (142 accessions), HapC (43 accessions), and HapD (158 accessions) alleles were of the TaWIR1b.2 allele. Seedling resistance identification showed that the HapB (142 accessions), HapC (43 accessions), and HapD (158 accessions), i.e., TaWIR1b.2, alleles, showed susceptibility to wheat powdery mildew. Figure 7 As shown in the figure, HapA, HapB, HapC, and HapD refer to the alleles of this gene on different materials. Among them, there are significant differences in powdery mildew resistance between the first genotype (HapA, which contains an alternative splicing site) and the latter three genotypes (HapB, HapC, and HapD, none of which contain an alternative splicing site).

[0104] Table 8: List of 480 Variety / Strain Materials

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] Example 5: VIGS silencing assay of TaWIR1b gene in different receptor materials and its phenotypic identification

[0111] The BSMV viral vector used for VIGS silencing consists of three parts: α, β, and γ. In this embodiment, the γ-WIR1b vector refers to the constructed γ vector that silences the TaWIR1b sequence. The γ vector refers to the wild-type vector without silencing function.

[0112] (1) Selection of receptor materials

[0113] Phenotypic identification of wheat powdery mildew seedling stage was conducted on the main wheat varieties in the Huang-Huai wheat region. Three materials with high susceptibility to wheat powdery mildew in the seedling stage, namely Zhoumai 18, XN18, and WM208, were selected as recipient materials for the VIGS experiment to further verify the function of the TaWIR1b gene.

[0114] (2) Construction of the TaWIR1b gene in a VIGS vector

[0115] Primers were designed based on the CDS sequence of the TaWIR1b gene shown in SEQ ID NO:2. Protective bases and restriction enzyme sites were added to the 5' ends of the forward and reverse primers, respectively, to form the upstream primer shown in SEQ ID NO:7: 5'-TTTTTTTAGCTAGCTGATTAATTAATCGCTCTCTTCGTCGCCA-3' and the downstream primer shown in SEQ ID NO:8: 5'-CTTCCGTTGCTAGCTGAGCGGCCGCCGTCGGAACAGCA GGGA-3'.

[0116] The VIGS silenced fragment of the TaWIR1b gene (SEQ ID NO:9) was amplified using this primer pair and then recovered for later use.

[0117] The γ vector was then digested with PacI and NotI, the γ linearized vector fragment was recovered, and the recombinant vector was constructed using homologous recombination to obtain the recombinant vector γ-WIR1b (i.e. BMSV-TaWIR1b).

[0118] (3) VIGS silencing assay of TaWIR1b gene in different materials

[0119] The BSMV viral vectors (α, β, γ) and the TaWIR1b gene silencing recombinant vector (γ-WIR1b) plasmids were linearized by restriction enzyme digestion. The restriction enzymes used were Mlu I (α, γ), Spe I (β), and BssHII (γ-WIR1b), respectively. The linearized plasmids were then transcribed in vitro using the RiboMAX™ Large Scale RNA Production Systems-T7 kit to obtain viral in vitro transcripts with different components. 2.5 μL each of the in vitro transcripts α, β, and γ / γ-WIR1b were mixed in a 1:1:1 ratio and diluted with an equal volume of DEPC water. 5 μL of the diluted mixture was added to 90 μL of FES buffer and thoroughly mixed. Three different treatments were set up for each experiment: a completely blank control group (WT), a viral blank control group (α+β+γ), and a gene silencing group (α+β+γ-WIR1b).

[0120] When infecting the plant with the virus, first spray a small amount of DEPC water onto the surface of the plant to be infected. Then, apply 8–10 μL of a LFES mixture to a clean glove and rub the second leaf of the seedling three times from the base to the tip of the leaf. Control the pressure during rubbing. After rubbing, spray a small amount of DEPC water from top to bottom to maintain humidity. Clean gloves must be changed for each treatment. After virus inoculation, the plant was placed in an incubator at 23±2℃ for 24 hours in a warm, humid, and dark environment. Then, the incubation period was adjusted to 16h / 8h light / dark cycle. Phenotypic changes were observed and recorded regularly.

[0121] (4) Phenotypic identification of TaWIR1b gene after VIGS silencing in different receptor materials

[0122] Two weeks after VIGS silencing, the relative expression of the TaWIR1b gene in silenced plants (BSMV-TaWIR1b) and virus-free vector plants (BSMV-γ0) was detected using qRT-PCR. The results are as follows: Figure 8 As shown, the results indicate that the relative expression level of the TaWIR1b gene was significantly downregulated in silenced plants of different recipient materials, and the gene was effectively silenced in silenced plants of different recipient materials (BSMV:Zhou18, BSMV:XN18, BSMV:WM208).

[0123] Further investigation into powdery mildew resistance in silenced plants from different recipient materials revealed that TaWIR1b gene-silenced plants exhibited significantly reduced powdery mildew spore attachment on leaf surfaces. Microscopic examination of the mycelium also showed delayed mycelial development and a significantly reduced number of mycelial colonies in TaWIR1b gene-silenced plants, indicating significantly enhanced disease resistance (see...). Figure 9 ).

[0124] In summary, silencing the TaWIR1b gene significantly enhanced plant resistance to wheat powdery mildew in different recipient materials, indicating that this gene is a negative regulatory gene in the wheat powdery mildew response process. Furthermore, silencing the TaWIR1b gene in different wheat varieties can improve the plant's resistance to wheat powdery mildew, demonstrating the broad applicability of TaWIR1b gene function manipulation.

[0125] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of the present invention should be included within the scope of the present invention.

Claims

1. Identification of wheat powdery mildew-related genes TaWIR1b The product is characterized by, The product is any one of the following: (1) The upstream primer with the nucleotide sequence shown in SEQ ID NO:4 and the downstream primer with the nucleotide sequence shown in SEQ ID NO:5; (2) A reagent or kit containing the primers described in (1); The wheat powdery mildew-related gene TaWIR1b The nucleotide sequence of the genome of the wheat variety is shown in SEQ ID NO:

1.

2. Identification of wheat powdery mildew-related genes TaWIR1b The composition of allele differential loci is characterized in that, include: The upstream primer with the nucleotide sequence shown in SEQ ID NO:4 and the downstream primer with the nucleotide sequence shown in SEQ ID NO:5; and differentially specific endonucleases EcoRII ; The wheat powdery mildew-related gene TaWIR1b The nucleotide sequence of the genome of the wheat variety is shown in SEQ ID NO:

1.

3. The use of the product of claim 1 or the composition of claim 2 in any of the following: (1) To identify or assist in the identification of wheat resistance to powdery mildew; (2) Breeding for resistance to wheat powdery mildew.

4. Detection of wheat powdery mildew-related genes TaWIR1b The method of allele typing is characterized by, Includes the following steps: Wheat genomic DNA was extracted as a template and PCR amplification was performed using the upstream primer with the nucleotide sequence shown in SEQ ID NO:4 and the downstream primer with the nucleotide sequence shown in SEQ ID NO:

5. This was followed by a specific endonuclease assay. EcoRII Perform single enzyme digestion on the amplified nucleotide fragments and perform the judgment as described in I or II below: Ⅰ: The amplified nucleotide fragments were processed EcoRII Wheat that yields 408 bp after enzyme digestion is TaWIR1b.1 The allele type was statistically significantly more resistant to wheat powdery mildew than wheat with a 263 bp fragment after enzyme digestion. II: The amplified nucleotide fragments were processed... EcoRII Wheat that has been digested with enzymes to a density of 263 bp is TaWIR1b.2 The allele type was statistically significantly less resistant to wheat powdery mildew than wheat whose enzyme digestion fragment was still 408 bp.

5. TaWIR1b Application of virus-induced silencing recombinant vectors in the breeding of wheat powdery mildew-resistant varieties and / or in the control of wheat powdery mildew, wherein the virus-induced silencing recombinant vectors contain genes. TaWIR1b The virus-induced silence of the nucleotide fragment; the gene TaWIR1b The full-length CDS nucleotide sequence shown in SEQ ID NO:2, or the gene described. TaWIR1b The nucleotide sequence of the genome of the wheat variety is shown in SEQ ID NO:1; the pathogen of powdery mildew is E. Bruchii powdery mildew E09.

6. The application according to claim 5, characterized in that, The method for constructing the virus-induced silencing recombinant vector includes the following steps: (1) Amplification using the upstream primer shown in SEQ ID NO:7 and the downstream primer with the nucleotide sequence shown in SEQ ID NO:

8. TaWIR1b Viral induction of silenced nucleotide fragments in genes and subsequent recovery: (2) The viral vector was double-digested with enzymes, and the recovered viral vector was then digested. TaWIR1b The virally silenced nucleotide fragment of the gene and the corresponding linearized viral vector fragment were used to construct the recombinant vector using homologous recombination.

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