Puccinia malvacearum-resistant gene pmaeu1 in aegilops tauschii and molecular markers and application thereof

Abstract

This invention relates to a powdery mildew resistance gene PmAeu1 derived from *Aegilops spp.*, its molecular markers, and applications, belonging to the field of genetic engineering. This invention utilizes the powdery mildew-resistant *Aegilops spp.* strain AE 1617 and the powdery mildew-susceptible strain AE 538 to construct mapping populations. Through BSR-Seq, fine genetic mapping, third-generation genome sequencing, and transgenic verification, a new powdery mildew resistance gene, PmAeu1, was cloned. This invention has demonstrated through transgenic wheat research that the PmAeu1 gene can significantly improve wheat resistance to powdery mildew. The *Aegilops spp.* powdery mildew resistance gene PmAeu1 can be applied to genetically improve wheat varieties to enhance wheat's powdery mildew resistance. This invention also develops a molecular marker, XAeuNLR1-2, for the PmAeu1 gene, which can be used to specifically detect the gene PmAeu1 as described in the claims and can be applied to marker-assisted breeding of the PmAeu1 gene.

Description

Technical Field

[0001] This invention relates to a gene PmAeu1 from Aegilops spp., its molecular markers, and applications, belonging to the field of genetic engineering. Background Technology

[0002] Wheat is a vital grain worldwide, with the largest planting area among all crops. In China, wheat is the second most important food crop after rice. During wheat cultivation, it is susceptible to various fungal infections, including powdery mildew, rust, and Fusarium head blight. Among these, wheat powdery mildew, caused by the grass-like powdery mildew fungus *Brucea fulvidraco*, is one of the most damaging foliar diseases in wheat production, typically reducing yield by 10-15%, and in severe cases, by up to 50%.

[0003] Aegilops scutellatus is a diploid wild relative of the Triticum genus, exhibiting resistance to various wheat pathogens and serving as an important source of disease-resistant genes for wheat improvement. To date, several resistance genes from Aegilops scutellatus have been successfully transferred into wheat, such as the powdery mildew resistance gene PmY39, leaf rust resistance genes Lr9 and Lr76, and stripe rust resistance gene Yr70.

[0004] Currently, with changes in the virulence of wheat pathogens, there is a predicament where major disease resistance genes lose their resistance. Therefore, it is urgent to explore and apply diverse disease resistance gene resources in order to continuously breed resistant wheat varieties. Summary of the Invention

[0005] The purpose of this invention is to overcome some problems existing in the prior art, especially the problem of the lack of existing resistance genes, and to provide a powdery mildew resistance gene PmAeu1 of Aegilops spp., its molecular marker and application.

[0006] This invention utilizes the powdery mildew-resistant Aegilops genus AE 1617 and the powdery mildew-susceptible Aegilops genus AE 538 to construct mapping populations. Through extreme pool transcriptome sequencing (BSR-Seq), fine genetic mapping, third-generation genome sequencing, and transgenic verification, a new powdery mildew resistance gene, PmAeu1, was cloned, and a corresponding diagnostic molecular marker was developed. This provides important genetic resources and a theoretical basis for breeding new powdery mildew-resistant wheat varieties.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0008] The present invention first provides a powdery mildew resistance gene PmAeu1, which is derived from Aegilops scutellariae. The DNA sequence of the gene is shown in SEQ ID No: 1, and the encoded amino acid sequence is shown in SEQ ID No: 2.

[0009] The present invention also provides biological materials containing the powdery mildew resistance gene PmAeu1, including recombinant expression vectors, recombinant engineered bacteria, etc.

[0010] The present invention also provides the application of the powdery mildew resistance gene PmAeu1 or biological materials containing the gene in improving wheat powdery mildew resistance; identifying, assisting in the identification or screening of wheat powdery mildew resistance; or breeding or assisting in the breeding of powdery mildew resistant wheat; or preparing products for identifying, assisting in the identification or screening of wheat powdery mildew resistance; or preparing products for wheat breeding or assisting in the breeding.

[0011] The present invention also provides a molecular marker XAeuNLR1-2, which is used to specifically detect the powdery mildew resistance gene PmAeu1. The primers for specifically amplifying the molecular marker include the upstream primer shown in SEQ ID No: 3 and the downstream primer shown in SEQ ID No: 4.

[0012] SEQ ID No: 3: agttc actgc tagag ttaga tgtc

[0013] SEQ ID No: 4: aggag gctag aagaa tatgc aga

[0014] The present invention also provides primers for detecting the powdery mildew resistance gene PmAeu1 or the molecular marker XAeuNLR1-2, said primers comprising an upstream primer as shown in SEQ ID No: 3 and a downstream primer as shown in SEQ ID No: 4.

[0015] The present invention also provides a reagent or kit comprising the primers.

[0016] The present invention also provides the application of the molecular marker, the primer, or the reagent or kit in detecting the gene PmAeu1 in crops, or screening or identifying crop resistance to powdery mildew, or in powdery mildew-resistant wheat breeding or assisted breeding.

[0017] The present invention also provides a method for detecting powdery mildew resistance, the method comprising: extracting DNA from the sample to be tested, performing PCR amplification using primers of the molecular marker, analyzing the amplification product, and if a 660bp fragment is obtained, the sample to be tested is a powdery mildew resistant sample.

[0018] This invention, through research on transgenic wheat, confirms that the PmAeu1 gene can significantly improve wheat resistance to powdery mildew. The powdery mildew resistance gene PmAeu1 from *Aegilops spp.* can be used to genetically improve wheat varieties to enhance powdery mildew resistance. This invention also developed the molecular marker XAeuNLR1-2 targeting the PmAeu1 gene sequence, which can be used to specifically detect the PmAeu1 gene as described in the claims and can be applied to marker-assisted breeding of the PmAeu1 gene.

[0019] The beneficial effects of this invention are:

[0020] (1) The gene sequence of PmAeu1, the powdery mildew resistance gene of Aegilops spp. provided in this invention, is reported for the first time both domestically and internationally.

[0021] (2) This invention utilizes a mapping population constructed from *Aegilops spp.* resistant and susceptible to powdery mildew. Through BSR-Seq analysis, fine genetic mapping, and third-generation genome sequencing and assembly, a powdery mildew-resistant gene, PmAeu1, was cloned from the powdery mildew-resistant *Aegilops spp.* AE 1617 line. PmAeu1 encodes a spiral-coil-nucleotide-binding-leucine-rich repeat (CC-NB-LRR, or NLR) resistance protein, with a sequence identity of less than 35% with the proteins encoded by previously reported powdery mildew-resistant genes such as Pm1 and Pm60. Transgenic analysis shows that transgenic wheat containing the PmAeu1 gene exhibits highly efficient powdery mildew resistance and has significant application value in wheat powdery mildew resistance breeding.

[0022] (3) Based on the DNA sequence of the PmAeu1 gene, this invention also developed a molecular marker XAeuNLR1-2, which can effectively track the PmAeu1 gene in wheat and can be applied to molecular marker-assisted breeding of the PmAeu1 gene. Attached Figure Description

[0023] Figure 1 The graph shows the reaction results of the Aegilops genus AE 1617 and AE 538 to powdery mildew.

[0024] Figure 2 The image shows the BSR-Seq analysis results of the powdery mildew resistance gene PmAeu1, with a significant peak located on the long arm (2UL) of chromosome 2U of Aegilops spp.

[0025] Figure 3 The results show the genetic map of PmAeu1 and the identification of the candidate gene AeuNLR1; among them, Figure 3 A represents the results of genetic analysis using 74 F3 disease-susceptible plants, with PmAeu1 located between molecular markers X2U-4 and X2U-6; Figure 3B shows the results of fine localization of PmAeu1 using 39 recombination events. PmAeu1 is located between the molecular markers X2U-4.4 and X2U-5B. The numbers below the bars represent the number of recombination events. Figure 3 C shows the results of identifying the candidate gene AeuNLR1 using BSR-Seq data (indicated by the box).

[0026] Figure 4 Electrophoresis images of Aegilops 'Small Umbrella' lines AE 538 and AE 1617 for the molecular marker XAeuNLR1; the arrow indicates the AeuNLR1-specific band, which is 293 bp in size.

[0027] Figure 5 A schematic diagram of the construction of the transgenic vector pLGY03-AeuNLR1; where AeuNLR1 is 13564bp in length and is divided into three fragments, A, B and C, which are sequentially ligated into the vector pLGY03. Fragment A is ligated through the restriction site BamHI / HindIII, while fragments B and C are ligated through the SmiI site (ATTTAAAT).

[0028] Figure 6 Results of detection of transgenic regenerated plants for molecular marker XAeuNLR1-2; T0#1-T0#6 are transgenic regenerated plants, the negative control is wheat Fielder, and the positive control is pLGY03-AeuNLR1 plasmid DNA; the arrow indicates the specific band of the AeuNLR1 / PmAeu1 gene, which is 660 bp in size.

[0029] Figure 7 The graph shows the response of transgenic regenerated plants to powdery mildew; T0#1-T0#6 are transgenic regenerated plants, and wheat Fielder is the negative control; + indicates XAeuNLR1-2 positive plants, and - indicates XAeuNLR1-2 negative plants.

[0030] Figure 8 Molecular marker XAeuNLR1-2 was used to detect different wheat varieties. M: DNA molecular weight marker DL2000; 1-12: different wheat varieties, in order: Chuanmai 42, Jimai 22, Jimai 23, Xumai 44, Yangmai 158, Yangmai 22, Yangmai 23, Zhongguochun, Zhenmai 13, Ningmai 13, Mianyang 11, and Kenong 199 (these wheat varieties are all well-known and commonly used); 13: positive control pLGY03-AeuNLR1 plasmid DNA; the arrow indicates the specific band of the AeuNLR1 / PmAeu1 gene, which is 660 bp in size. Detailed Implementation

[0031] To enable those skilled in the art to better understand the technical solutions of the present invention, the preferred embodiments of the present invention will be described in detail below. However, the following embodiments do not limit the scope of protection of the present invention.

[0032] In the embodiments of the present invention, unless otherwise described, conventional experimental methods were used. The processes involved in the embodiments are all understandable and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field. Unless otherwise specified, the reagents and materials involved are all obtained through commercial means.

[0033] Example 1. Genetic mapping of the powdery mildew resistance gene PmAeu1 in Aegilops spp.

[0034] This invention utilizes the powdery mildew strain BgtYZ01 (a known strain) to identify powdery mildew in *Aegilops spp.* It found that the *Aegilops spp.* strain AE 1617 (a known strain, sourced from the German Germplasm Bank: https: / / gbis.ipk-gatersleben.de / gbis2i) exhibits powdery mildew resistance, while AE 538 (a known strain, sourced from the German Germplasm Bank: https: / / gbis.ipk-gatersleben.de / gbis2i) is susceptible to powdery mildew (e.g., ...). Figure 1 (As shown).

[0035] The resistant AE 1617 and the susceptible AE 538 were crossed and multiplied to obtain the F2 population. 253 F2 plants were inoculated with powdery mildew strain BgtYZ01 (a known strain) and cultured at 22°C for 8 days. Powdery mildew disease incidence was then assessed. The number of resistant and susceptible plants was 194 and 59 respectively, consistent with a Mendelian genetic ratio of 3:1 (χ²). 2 =0.381, P=0.537), indicating that the resistance of AE 1617 is controlled by a dominant gene. Powdery mildew was identified in 1020 individual plants from 8 F3 families. 774 resistant plants and 246 susceptible plants were found, consistent with the theoretical ratio of 3:1 (χ²=0.381, P=0.537). 2 =0.424, P=0.515). This powdery mildew resistance gene in the Aegilops spp. AE 1617 is named PmAeu1.

[0036] Fifty resistant plants from the F3 family in step (1) were used to construct an resistance pool, and 50 susceptible plants were used to construct a susceptibility pool. High-throughput RNA sequencing was performed using BSR-Seq technology, with *Aegilops spp.* TA1851 (NCBI accession number GCA032464435.1) as the reference genome. This revealed 18,576 SNPs and 9,242 InDels, of which 2,423 SNPs and 1,356 InDels were located on the long arm (2UL) of chromosome 2U of *Aegilops spp.*, indicating that the gene PmAeu1 is located on chromosome 2UL of *Aegilops spp.* (e.g., ...). Figure 2 (As shown).

[0037] Based on the SNPs and InDels near the significant peaks in BSR-Seq analysis, five pairs of competitive allele-specific PCR (KASP) markers and two pairs of cut-amplified polymorphic sequence (CAPS) markers (Table 1) were designed, spanning a 17.5 Mb interval. Genotypic analysis was performed on 74 F3 susceptible plants. Combined with powdery mildew phenotypic data, the gene PmAeu1 was located in a 6.4 Mb interval between molecular markers X2U-4 and X2U-6 on chromosome 2UL. This region contains 19 recombination events (such as...). Figure 3 A). The population was further expanded, and 20 recombination events were screened from 172 F3 infected plants. Sanger sequencing was then used to genotype the 39 recombination events obtained before and after the screening, locating the PmAeu1 gene to a 326 kb interval between X2U-4.4 and X2U-5B in the TA1851 reference genome (e.g., ...). Figure 3 B).

[0038] The KASP marker analysis method: A 20 μL reaction mixture contained 1×KASP Master Mix (LGC, Teddington, UK), 0.2 μM F, V, and R primers, and 50 ng of genomic DNA. The PCR reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, with the temperature decreasing from 64℃ to 58℃ (0.6℃ decrease per cycle), for 10 cycles; 94℃ denaturation for 20 s, extension at 58℃ for 60 s, for 38 cycles. PCR amplification and fluorescence signal recording were performed using an Applied Biosystems QuantStudio 3 real-time PCR instrument (Thermo Fisher Scientific, USA).

[0039] CAPS labeling analysis method: A 25 μL reaction mixture contained approximately 50 ng of genomic DNA, 1×PCR buffer, 200 mM dNTPs, two primers at a final concentration of 2 μM each, and 1 U Taq DNA polymerase. The mixture was brought to 25 μL with sterile distilled water. The PCR program was as follows: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 20 seconds, 55℃-60℃ annealing for 30 seconds, 72℃ extension for 60 seconds, 35 cycles; 72℃ extension for 5 minutes; storage at 4℃. PCR products were separated by electrophoresis on an 8% non-denaturing polyacrylamide gel and stained with silver.

[0040] Table 1. Polymorphic molecular markers used for PmAeu1 genetic mapping

[0041]

[0042]

[0043] Note: KAPS, competitive allele-specific PCR; CAPS, cleavage and amplification of polymorphic sequences, NcoI and PstI are the restriction endonucleases used in CAPS; SS, Sanger sequencing; STS, sequence tag site.

[0044] Example 2. Cloning and Transgenic Analysis of the PmAeu1 Gene for Powdery Mildew Resistance in Aegilops spp.

[0045] This invention utilizes PacBio HiFi long-read sequencing of the genome of the Aegilops spp. AE 1617, a strain resistant to powdery mildew, yielding approximately 57.2 Gb of HiFi reads, achieving about 13.5 times the coverage of the TA1851 genome (4.25 Gb). The Aegilops spp. AE 1617 genome data was assembled to obtain 2,211 fragments (N50 = 5.43 Mb, total length = 4.29 Gb). In the sequenced genome, the molecular markers X2U-4.4 and X2U-5B correspond to a 287 kb fragment. Using the obtained AE 1617 genome as a reference, BSR-Seq data mapping analysis revealed a candidate gene, AeuNLR1, encoding a CC-NBS-LRR (NLR) class resistance protein, which appeared only in resistant strains but not in susceptible strains (e.g., ...). Figure 3 C).

[0046] A molecular marker, XAeuNLR1, was developed targeting the candidate gene AeuNLR1. PCR amplification using XAeuNLR1 further confirmed the presence of the AeuNLR1 gene (with a specific 293 bp product) in the diploid powdery mildew-resistant *Aegilops spp.* AE 1617, while the AeuNLR1 gene was absent in the powdery mildew-susceptible *Aegilops spp.* AE 538. Figure 4The full-length AeuNLR1 gene, 13,564 bp, was obtained. The specific sequence is shown in SEQ ID No: 1. The complete coding region is 4737 bp (from position 3234 to position 7970), encoding a protein of 1578 amino acids. The amino acid sequence is shown in SEQ ID No: 2. It belongs to the CC-NBS-LRR (NLR) class of disease-resistant proteins.

[0047] A 13564 bp genomic DNA fragment containing the promoter, coding region, and terminator of the AeuNLR1 gene was divided into three fragments, A, B, and C (each fragment had a 20 bp overlap, serving as homologous arms for cloning). The three fragments were amplified by PCR using primers AeuNLR1-P1 / P2, AeuNLR1-P3 / P4, and AeuNLR1-P5 / P6 (Table 2), respectively. High-fidelity PrimeSTAR GXL DNA polymerase was used for PCR amplification. The PCR reaction system consisted of approximately 50 ng template DNA, 1×PrimeSTAR GXL buffer, 200 μM dNTP Mixture, two primers at a final concentration of 0.2 μM each, and 1.25 U PrimeSTAR GXL DNA polymerase. The reaction system was brought to 50 μL with sterile distilled water. The PCR reaction program was as follows: 98℃ pre-denaturation for 3 minutes; 98℃ denaturation for 10 seconds, 55℃ annealing for 15 seconds, 68℃ extension for 3 minutes, 35 cycles; 68℃ extension for 5 minutes; store at 4℃.

[0048] PCR amplification products were separated by electrophoresis on a 1.2% agarose gel and stained with nucleic acid dyes. Gene fragments A, B, and C were independently amplified from the AE 1617 genomic DNA using gel purification technology. Fragments A and B each contain a Smithi restriction site (ATTTAAAT), introduced from the reverse primers (AeuNLR1-P2 and AeuNLR1-P4). The pLGY03 vector (publicly available, provided by the Shandong Academy of Agricultural Sciences) was digested with HindIII / BamHI to remove the original expression cassette and linearize the plasmid DNA. Fragment A (4,657 bp) was then ligated into the HindIII / BamHI-digested pLGY03 using the ClonExpress Ultra one-step cloning kit to form the recombinant vector pLGY03-A-SmiI. Next, fragment B (4561 bp) was ligated into the Smithi-digested pLGY03-A-SmiI using the same kit to form the recombinant vector pLGY03-AB-SmiI. Finally, fragment C (4346 bp) was inserted into the Smithi-digested pLGY03-AB-SmiI to generate pLGY03-ABC, which is pLGY03-AeuNLR1. Figure 5).

[0049] Sanger sequencing was performed on the recombinant vector pLGY03-AeuNLR1 using primers AeuNLR1-P7-AeuNLR1-P26 (Table 2). The sequence was confirmed to be completely identical to the original sequence of the AeuNLR1 gene, SEQ ID No:1, indicating successful construction of the recombinant vector. Then, Agrobacterium-mediated transformation was used to transform the recombinant vector pLGY03-AeuNLR1 into the powdery mildew-susceptible wheat variety Fielder (publicly known and provided by the Shandong Academy of Agricultural Sciences), yielding six T0 regenerated plants. Since the molecular marker XAeuNLR1 exhibits non-specific amplification products in the hexaploid wheat background, making it unsuitable for effective detection of transgenic components, this invention redeveloped a molecular marker XAeuNLR1-2 based on the AeuNLR1 gene sequence SEQ ID No:1. The upstream and downstream primer sequences are shown in SEQ ID No:3 (based on gene sequence positions 7872 to 7895) and SEQ ID No:4 (based on gene sequence positions 8509 to 8531), respectively. PCR detection of T0 regenerated plants using XAeuNLR1-2 revealed that 5 plants contained a specific 660bp band (from position 7872 to 8531 in the gene sequence), indicating the presence of the AeuNLR1 gene. One plant did not show the 660bp specific band, indicating the absence of the AeuNLR1 gene. Figure 6 Subsequently, these regenerated plants were inoculated with the powdery mildew strain BgtYZ01. It was found that all five AeuNLR1-positive plants were resistant, while one AeuNLR1-negative plant and the control, Fielder, were susceptible. Figure 7 This confirms that the gene AeuNLR1 at the PmAeu1 locus has anti-powdery mildew function, proving that AeuNLR1 is the target gene PmAeu1 screened in Example 1 of this invention.

[0050] The sequence information for SEQ ID No:3 and SEQ ID No:4 is shown below:

[0051] SEQ ID No: 3: agttc actgc tagag ttaga tgtc,

[0052] SEQ ID No: 4: aggag gctag aagaa tatgc aga.

[0053] Table 2. Primers for cloning and sequencing of the PmAeu1 gene

[0054]

[0055]

[0056] The sequence information for SEQ ID No: 1 is as follows:

[0057]

[0058] The sequence of SEQ ID No:2 is as follows:

[0059] Met Glu Thr Ala Gly Ile Arg Ala Ala Ser Trp Val Val Gly Lys Ala LeuSer Pro Leu Ser Gly Gly

[0060] Leu Leu Glu Ala Trp Gly Ala Ser Ser Met Leu Gly Ser Asn Met Glu AlaLeu Lys Met Gln Leu Leu

[0061] Tyr Ala Gln Ala Met Leu Ser Asn Val Arg Gly Arg Glu Ile His Asn ProAla Leu Gly Glu Leu Leu

[0062] Asp Lys Leu Arg Gln Leu Ala Tyr Arg Ala Glu Asp Val Leu Asp Glu LeuAsp Tyr Phe Arg Ile Gln

[0063] Asp Glu Leu Asp Gly Thr Tyr His Ala Ala Asp Ala His Ala Ala Gly CysVal Gln Asp Leu Ala Leu

[0064] Asn Ala Arg His Thr Ala Arg Ala Cys Val Asn Lys Leu Lys Leu Pro ValCys Ser His Ala Thr Lys

[0065] His Asp Lys Gln Asp Asp Gly Asp Lys Glu Arg Cys Leu Ser Gly Leu ArgPhe Cys Gly Gly Arg Glu

[0066] Ile Ser Ser Ser Pro Pro Ser Arg Ala Asn Gln Val Val Gln Ala Val HisGly Gly Cys Ile Pro Lys Val

[0067] Ala Ser Ser Ala Arg Glu Ala Ala His Ala Ile Gly Lys Asn Phe Pro CysTyr Ser Phe Ser Ser Ala Arg

[0068] Asn Asp Asp His Asn Ile Gly Met Ser Lys Ser Ser Asn Val Ser Gln AsnGly Arg Arg Phe Leu Cys

[0069] Gly Ala Trp Leu Ser Pro Lys Ala Pro Glu Lys Ser His Ala Ile Gln AlaArg lys Leu Lys Phe Asp Arg

[0070] Val Glu Met Ser Thr Lys Ile Lys Asp Ile Ile Glu Glu Phe Lys Pro IleCys Gly Met Val Ser Thr Ile

[0071] Leu Ser Leu Glu Leu Leu Gly Ser Arg His Thr Pro Gly Gln Glu Glu ThrAla Met Asn Arg Val Lys

[0072] Thr Thr Pro Glu Ile Val Glu Pro Lys Leu Tyr Gly Arg Asp Ser Gln ThrLys Ile Val Val Asp Glu Ile

[0073] Ile Thr Gly Glu Cys Cys Glu Leu Thr Val Leu Pro Ile Val Gly Met GlyGly Ile Gly Lys Thr Thr Phe

[0074] Thr Arg His Ile Tyr Glu Lys Met Lys Ser His Phe Gln Ile Pro Ile TrpVal Cys Val Ser Phe Asp Phe

[0075] Asn Ala Asn Arg Leu Ala Lys Asp Ile Met Glu Lys Ile Pro Gln Ile AspLys Glu Glu Lys Tyr Trp Ser

[0076] Asp Glu Glu Arg Ile Met Arg Arg Ile Lys Gly Lys Arg Val Leu Leu ValLeu Asp Asp Val Trp Thr

[0077] His His Glu Asn Glu Trp Gly Lys Leu Leu Asn Leu Phe Arg ly sAsp GlyAla Lys Gly Asn Met Val

[0078] Ile Val Thr Thr Arg Ile Pro Glu Val Ala Asn Thr Val Lys Thr Thr LysCys Ser Leu Gln Leu Glu His

[0079] Leu Cys Pro Glu Asp Phe Met Tyr Phe Phe Glu Glu Cys Val Phe Gly AspGln Lys Pro Trp Val Asp

[0080] His Pro Glu Leu Ser Glu Val Gly Lys Lys Ile Val Asp Lys Leu Lys GlySer Pro Leu Ala Ala Lys Thr

[0081] Val Gly Arg Leu Leu Arg Asn Lys Leu Thr Leu Asn His Trp Thr Ser ValLeu Glu Ser Lys Glu Trp

[0082] Glu Ser Gln Ile Asn Glu Asn Asp Ile Met Pro Ala Leu Lys Leu Ser TyrAsp Tyr Leu Pro Phe His

[0083] Leu Gln Gln Cys Phe Ser Tyr Cys Ala Leu Phe Pro Glu Asp Tyr Glu PheGly Ser Gln Glu Leu Val

[0084] His Leu Trp Ile Gly Leu Asp Ile Leu His Ser Cys Asp Gln Asn Arg lysArg Ile Glu Asp Val Gly Leu

[0085] Cys Tyr Ile Asn Asp Leu Val Asn His Gly Phe Leu Lys Leu Asn Lys MetVal Asp Gly Cys Pro Tyr

[0086] Tyr Ile Ile His Asp Leu Leu His Glu Leu Ala Val Lys Val Ser Ser TyrGlu Cys Leu Ser Ile Tyr Ser

[0087] Ser Asn Met Arg His Ile Gln Ile Pro Thr Ser Val Arg His Leu Ser IleIle Val Asp Asn Thr Asp Val

[0088] Lys Asp Arg Met Ser Phe Asp Asp Tyr Ala Gly Asn Leu Ser Ala Leu AspLys Arg Leu Lys Val Glu

[0089] Gln Leu Arg Thr Leu Met Leu Phe Gly Glu Tyr His Gly Ser Phe Ala GlnThr Phe Gly Gly Leu Phe

[0090] Ser Lys Ala Thr Ala Leu Arg Val Ile Phe Leu Ser Glu Pro Ser Tyr AsnLeu Asp Asp Ile Leu His Asn

[0091] Phe Ser Lys Leu Val His Leu Arg Tyr Leu Arg Ile Lys Ser Ser Ser TyrHis Arg Gly Phe Cys Leu Pro

[0092] Ser Glu Leu Phe Arg Leu Tyr His Leu Glu Val Ile Asp Leu Gln Lys CysPhe Val Ile Ser Ser Ser Thr

[0093] Arg Asn Ile Gly Asn Leu Val Lys Leu His His Phe Leu Val Pro Ala TyrGly Leu Arg Phe His Leu

[0094] Asp Ile Tyr Gly Val Gly Lys Leu Lys Phe Leu Gln Glu Leu Arg Ala PheArg Val Gly Lys Glu Ser

[0095] Asp Gly Phe Glu Leu Ser Gln Leu Gly Gln Leu Thr Glu Ile Gly Gly LeuLeu Gly Ile Tyr Asn Leu

[0096] Asp Asn Val Gln Thr Lys Glu Glu Ala Asn Glu Ser Lys Leu Met His LysAsn His Leu Arg Glu Leu

[0097] Ile Leu Gly Trp Asp Ala Thr Arg Ser Asn Lys Asp Pro Val Gln Glu GluAsn Val Leu Glu Asn Leu

[0098] Val Pro His Arg Asn Leu Gln Lys Leu His Ile Arg Gly His Gly Gly ThrArg Cys Pro Ala Trp Leu

[0099] Arg Thr Asn Leu Ser Val Lys Cys Leu Glu Ser Leu Ser Leu Asp Gly ValSer Trp Lys Asn Leu Pro

[0100] Pro Leu Gly Asp Met Trp Met Val Asn Glu Leu Gly Glu Glu Tyr Gln CysCys Ser Ile Ser Pro Pro

[0101] Gly Phe His Asn Leu Lys Arg Leu Glu Leu Arg Ser Ile Ser Arg Leu LysLys Trp Val Gly Asn Gly

[0102] Ala Cys Pro Phe Phe Ser His Leu Glu Val Leu Ile Ile Arg Gly Cys SerGlu Leu Ile Glu Leu Pro Phe

[0103] Ser His Pro Pro Thr Cys Ser Gln Ala Gln Trp Glu Glu Lys Ile Gly TrpPhe Pro Lys Leu Arg Glu

[0104] Leu Glu Ile Ala Asp Cys Pro Lys Leu Ala Ser Leu Leu Pro Ile Pro TrpArg Thr Gly Ala Pro Cys Ser

[0105] Ala Gln Ile Ala Arg Val Gly Ser Gly Phe Glu Glu Ile Val Tyr Pro LysLys Tyr Gly Ser Glu Leu Ser

[0106] Leu Lys Ile Glu Gly Lys Asp Gly Gln Ala Glu Val Phe Trp Ser Gly LeuAsn Phe Ser Asn Leu Ala

[0107] Asp Leu Glu Glu Leu Phe Met Lys Arg Ile Pro Pro Leu Pro Leu Asp AspPhe Gln Val Leu Thr Ser

[0108] Leu Lys Lys Ile Val Ile Glu Gly Ala Gly Asn Ile Leu Pro Leu Val GluGly Asp Arg Cys His Gly Met

[0109] Tyr Arg Phe Pro Val Glu Asp Leu Glu Leu Ser His Cys Asp Ala Gly GlyLys Glu Leu Thr Leu Leu

[0110] Leu Ser Phe Leu Pro Asn Leu Ser Lys Leu Lys Ile Met Ser Cys Asn AsnLeu Thr Gly Leu Ala Val

[0111] Val Glu Asp Ala Glu Thr Val Phe Arg Gly Gln Glu Gln Gln Thr Arg ValGly Glu Glu Glu Ile Ile Thr

[0112] Ala Glu Val Ala Ala Glu Leu Gly Leu Leu Leu Leu Pro Pro His Leu HisGlu Leu Ser Phe Trp Asn

[0113] Cys Pro Lys Leu Ser Met Leu Pro Asn Pro Leu His Asp Asp Gln Thr GlnThr Gly Thr Ser Thr Gly

[0114] Arg Arg Gly Gly Gly Leu Gln Arg Leu His Ser Ile Arg Leu Leu Ala ValSer Asn Cys Pro Glu Phe

[0115] Leu Ser Phe Tyr Ser Ser Ser Ser Ser Ser Ser Phe Pro Phe Pro Thr CysLeu Gln Asp Leu Thr Leu

[0116] Gln Asp Met Lys His Val His Thr Leu Gln Ala Leu Ser Asn Leu Thr SerLeu Thr Glu Leu Arg Leu

[0117] Arg Gly Leu Gly Asn Ser Arg Asn Glu Gly Leu Trp Pro Leu Leu Ala HisGly Gln Leu Ala Lys Leu

[0118] Thr Leu His Asn Glu Ser Asp Phe Phe Ala Gly Ser Asp Pro Ser Arg LeuHis Asp Lys Glu Val Phe

[0119] Ser Arg Ser Ser Lys Leu Phe Asp Leu Ser Thr Thr Asn Asn Thr Gly ValLeu Ala Ala Pro Ile Cys

[0120] Gly Leu Leu Ser Ser Thr Leu Thr Arg Phe Glu Leu His Cys Glu Asp GluVal Glu Cys Leu Thr Lys

[0121] Glu Gln Glu Val Ala Leu His Leu Leu Ile Asn Leu Arg Gln Leu Val PheTrp Thr Gly Arg lys Leu

[0122] Arg His Leu Pro Ala Gly Leu His Asn Leu Ile Ser Leu Lys Lys Leu GluIle Leu Trp Cys Phe Ala Ile

[0123] Gln Ser Leu Pro Ser Leu Pro Thr Ser Leu Gln Glu Leu Glu Ile His GluCys Arg Ala Ile Glu Ser Leu

[0124] Pro Asn Asn Leu Pro Gly Ser Leu Glu Lys Leu Lys Ile Ser Ala Cys PheAla Ile Lys Ser Leu Leu Lys

[0125] Asp Gly Leu Pro Ser Ser Leu Leu Glu Leu Asp Val His Tyr Ala Asn SerGlu Glu Leu Lys Arg Asp

[0126] Cys Arg lys Phe Ile Gly Thr Ile Pro Ile Val Arg Thr

[0127] Example 3. Development of XAeuNLR1-2, a diagnostic molecular marker for the powdery mildew resistance gene PmAeu1 in Aegilops spp.

[0128] This invention developed a molecular marker XAeuNLR1-2 targeting the sequence SEQ ID No:1 of the AeuNLR1 / PmAeu1 gene. The upstream and downstream primer sequences are shown in SEQ ID No:3 and SEQ ID No:4, respectively. The molecular marker XAeuNLR1-2 was used to perform PCR amplification on six transgenic T0 regenerated plants from the wheat variety Fielder in Example 2.

[0129] The PCR reaction system consisted of approximately 50 ng template DNA, 1×PCR buffer, 1.5 mM MgCl2, 200 mM dNTPs, two primers at a final concentration of 0.2 μM each, and 1 U Taq DNA polymerase. The reaction system was then replenished to 25 μL with sterile distilled water.

[0130] The PCR reaction program was as follows: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 20 seconds, 55℃-60℃ annealing for 30 seconds, 72℃ extension for 60 seconds, 35 cycles; 72℃ extension for 5 minutes; storage at 4℃. PCR amplification products were separated by electrophoresis on a 1.2% agarose gel and stained with nucleic acid dyes.

[0131] A 660bp band targeting the PmAeu1 gene was found in all five powdery mildew-resistant T0 regenerated plants, while this specific band was not found in one powdery mildew-susceptible plant. Figure 6 This indicates that the molecular marker XAeuNLR1-2 can specifically track the PmAeu1 gene in the wheat background, and has potential application value for molecular marker-assisted breeding of the PmAeu1 gene derived from Aegilops spp.

[0132] Using the same PCR conditions and electrophoresis detection method, the molecular marker XAeuNLR1-2 was used to detect different wheat varieties (including: Chuanmai 42, Jimai 22, Jimai 23, Xumai 44, Yangmai 158, Yangmai 22, Yangmai 23, Zhongguochun, Zhenmai 13, Ningmai 13, Mianyang 11, Kenong 199, etc., all of which are publicly known materials) that do not contain the genomic components of *Aegilops spp.* Except for the positive control pLGY03-AeuNLR1 plasmid DNA, which showed a specific band (660 bp) of the AeuNLR1 / PmAeu1 gene, no other wheat varieties showed this specific band. Figure 8 This indicates that the molecular marker XAeuNLR1-2 did not exhibit any interfering bands in these wheat varieties, suggesting that the molecular marker XAeuNLR1-2 is valuable for marker-assisted selection breeding of the PmAeu1 gene.

Claims

1. A powdery mildew resistance gene PmAeu1, characterized in that, The gene is derived from Aegilops spp., and the nucleotide sequence of the gene is shown in SEQ ID No:

1.

2. A biological material containing the powdery mildew resistance gene PmAeu1 as described in claim 1, wherein the biological material is a recombinant expression vector or a recombinant engineered bacterium.

3. The protein encoded by the powdery mildew resistance gene PmAeu1 according to claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID No:

2.

4. The application of the powdery mildew resistance gene PmAeu1 of claim 1 or the biological material of claim 2 in improving wheat powdery mildew resistance; identifying, assisting in the identification or screening of wheat powdery mildew resistance; or breeding or assisting in the breeding of powdery mildew-resistant wheat; or preparing products for identifying, assisting in the identification or screening of wheat powdery mildew resistance; or preparing products for wheat breeding or assisting in the breeding.

5. Molecular marker XAeuNLR1-2, characterized in that, The molecular marker is used to specifically detect the powdery mildew resistance gene PmAeu1 of claim 1 in wheat, and the primers used to specifically amplify the molecular marker are the upstream primer shown in SEQ ID No: 3 and the downstream primer shown in SEQ ID No:

4.

6. Primers for detecting the powdery mildew resistance gene PmAeu1 as described in claim 1 or the molecular marker XAeuNLR1-2 as described in claim 5, characterized in that, The primers are the upstream primer shown in SEQ ID No: 3 and the downstream primer shown in SEQ ID No:

4.

7. A reagent or kit, characterized in that, The reagent or kit contains the primers of claim 6.

8. The application of the molecular marker XAeuNLR1-2 as described in claim 5, or the primer as described in claim 6, or the reagent or kit as described in claim 7 in detecting the PmAeu1 gene in wheat, or screening or identifying wheat resistance to powdery mildew, or in breeding or assisted breeding of powdery mildew-resistant wheat.

9. A method for detecting wheat powdery mildew resistance, characterized in that, The method includes: extracting DNA from the sample to be tested, performing PCR amplification using primers for the molecular marker described in claim 5, analyzing the amplification product, and if a 660 bp fragment is obtained, then the sample to be tested is a powdery mildew resistant sample.

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