Cloning and functional marker development for wheat hybrid necrosis gene Ne1

Abstract

The application discloses cloning of a wheat hybridization necrosis gene Ne1 and development of a functional marker. The application provides a protein consisting of an amino acid sequence shown in SEQ ID No. 3. A nucleic acid molecule encoding the protein is also provided, and is named as a Ne1 gene, which is a DNA molecule shown in SEQ ID No. 2, or a DNA molecule shown in SEQ ID No. 1 or 2407-10,563 of SEQ ID No. 1. The application provides gene location, map-based cloning, mutant and transgenic function verification and development of a functional marker of the wheat hybridization necrosis gene Ne1. Cloning of the wheat hybridization necrosis gene Ne1 can completely reveal the mystery of wheat hybrid necrosis formation, can provide theoretical guidance for overcoming genetic obstacles of hybridization necrosis in the process of wheat hybridization breeding, can promote cultivation of new wheat varieties with high efficiency and excellent traits, and has important significance.

Description

Technical Field

[0001] This invention belongs to the fields of crop molecular biology and molecular breeding, specifically involving the cloning and functional marker development of the wheat hybrid necrosis gene Ne1. Background Technology

[0002] Wheat is an important food crop, with about one-third of the world's population relying on it as their staple food. Hybrid breeding is currently the most common and effective breeding method for developing new wheat varieties. However, in the process of wheat hybrid breeding, hybrid necrosis often occurs in the offspring, which greatly limits the free aggregation of superior traits from the parents (Caldwell et al., 1943; Hermsen 1963a), seriously hindering the genetic improvement of wheat and the selection of new varieties.

[0003] Hybrid necrosis in wheat is controlled by a pair of complementary genes, Ne1 and Ne2 (Chu et al., 2006), which aggregate through hybridization, causing hybrid necrosis. Although wheat hybrid necrosis has been observed for 100 years (Sax 1921), its molecular mechanism remains unclear, making it a hot and challenging area of ​​research both domestically and internationally. Recently, several research teams at home and abroad reported cloning one of the hybrid necrosis genes, Ne2, which encodes a CC-NBS-LRR type disease resistance protein and demonstrated that it is the same gene as the wheat high-temperature resistance to leaf rust gene Lr13 (Yan et al., 2021; Hewitt et al., 2021; Si et al., 2021b).

[0004] Nishikawa et al. (1974) located the Ne1 gene at 5BL, 9.4 ± 1.5 cM from the centromere, using common wheat telomeres (Nishikawa et al., 1974). Chu et al. (2006) constructed genetic linkage maps of Ne1 and Ne2 using SSR molecular markers, showing genetic distances of 2.0 cM and 3.2 cM between Ne1 and Ne2 and their nearest SSR markers Xbarc74 and Xbarc55, respectively (Chu et al., 2006). Recently, fine mapping of the Ne1 gene has also made progress. Li et al. (2021) located the Ne1 gene at a genetic distance of 0.19 cM between molecular markers Xwgrc3074 and Xwgrc3009, corresponding to a physical distance of 4.45 Mb in Chinese spring (Li et al., 2021). Si et al. (2021) located the Ne1 gene between molecular markers 5B-383 and SN-2142, corresponding to a physical distance of 4.06 Mb from the Chinese spring (Si et al., 2021). Zhang et al. (2022) reported that the Ne1 gene was finely mapped to a distance of 0.5 cM between molecular markers Nwu_5B_4137 and Nwu_5B_5114 (Zhang et al., 2022).

[0005] However, the Ne1 gene has not yet been cloned. Cloning the Ne1 gene and using the unique genetic system of Ne1-Ne2 interaction to elucidate the molecular mechanism of hybrid necrosis formation will completely unveil the mystery of wheat hybrid necrosis. It will also provide theoretical guidance for overcoming the genetic barriers to hybrid necrosis in wheat hybridization breeding, and lay the theoretical foundation for precise molecular design of disease resistance using the NLR resistance protein encoded by Ne2. This is of great significance for breeding high-yielding, disease-resistant, and superior new wheat varieties. Summary of the Invention

[0006] The purpose of this invention is to provide the cloning and functional marker development of the wheat hybrid necrosis gene Ne1.

[0007] In a first aspect, the present invention provides a protein, named Ne1 protein, which is any of the proteins shown in A1)-A4) below:

[0008] A1) A protein consisting of the amino acid sequence shown in SEQ ID No. 3;

[0009] A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 3;

[0010] A3) A protein having the same function as the amino acid sequence shown in SEQ ID No. 3, with one or more amino acid residues substituted and / or deleted and / or added;

[0011] Proteins that have 99%, 95%, 90%, 85%, or 80% homology and the same function of any of the amino acid sequences defined in A4 and A1-A3).

[0012] In a second aspect, the present invention provides a nucleic acid molecule encoding the protein described in the first aspect.

[0013] The nucleic acid molecule mentioned above is named gene Ne1, located on wheat chromosome 5BL, and can be any of the following:

[0014] B1) The DNA molecule shown in SEQ ID No. 2 is a cDNA sequence;

[0015] B2) The DNA molecule shown in SEQ ID No. 1 or positions 2407-10,563 (genomic sequence) of SEQ ID No. 1;

[0016] B3) DNA molecules that have more than 98% homology with the DNA sequence defined by B1) or B2) and encode the same functional protein;

[0017] B4) DNA molecules that hybridize with DNA sequences defined by B1) or B2) under strict conditions and encode the same functional protein;

[0018] DNA molecules that share more than 90% homology with DNA sequences defined by B5 and B1 or B2 and encode the same functional protein.

[0019] Thirdly, the present invention provides expression cassettes, recombinant vectors, or recombinant microorganisms containing the nucleic acid molecules described in the second aspect.

[0020] Fourthly, the present invention provides the use of the protein described in the first aspect, the nucleic acid molecule described in the second aspect, or the expression cassette, recombinant vector, or recombinant microorganism described in the third aspect in any of the following:

[0021] C1) Cultivate plants with a necrotic phenotype, wherein the plants contain the Ne2 gene;

[0022] C2) Induces plants containing the Ne2 gene to exhibit a necrotic phenotype;

[0023] The C3 gene, in conjunction with the Ne2 gene, causes plant necrosis.

[0024] Fifthly, the present invention provides a method for preparing a transgenic plant with a necrosis phenotype, comprising the following steps: introducing the nucleic acid molecule described in the second aspect into a target plant containing the Ne2 gene to obtain a transgenic plant with a necrosis phenotype.

[0025] In a sixth aspect, the present invention provides primer pairs for amplifying the full length or a portion of the nucleic acid molecule described in the second aspect, which consist of a single-stranded DNA molecule shown in SEQ ID No. 4 and a single-stranded DNA molecule shown in SEQ ID No. 5.

[0026] In a seventh aspect, the present invention provides PCR reagents or kits containing the primer pairs described in the sixth aspect.

[0027] Eighthly, the present invention provides the use of the primer pair or the PCR reagent or kit described in the seventh aspect in identifying whether a plant variety contains the nucleic acid molecule described in the second aspect.

[0028] In the above text, the plant variety can be a hybrid offspring containing the Ne1 and Ne2 genes, and more specifically, a hybrid offspring of the necrotic line M114. In the embodiments of the present invention, it is a hybrid offspring of the necrotic line M114 and the common wheat variety Zhoumai 22.

[0029] The plants mentioned above include, but are not limited to, wheat.

[0030] The EMS mutagenesis used in this invention to create the necrotic line M114 regreen mutant has the following phenotype: Figure 4 As shown in the figure.

[0031] This invention provides a transgenic vector driven by the self-promoter of the Ne1 gene, which is constructed using the method reported in the reference (Lu et al. A rare gain of function mutation in a wheat tandem kinase confersresistance to powdery mildew. Nat. Commun. 2020; 11, 680).

[0032] This invention provides gene localization, map-based cloning, mutant and transgenic functional verification of the wheat hybrid necrosis gene Ne1, as well as the development of functional markers. Cloning the wheat hybrid necrosis gene Ne1 will completely unveil the mystery of wheat hybrid necrosis formation, providing theoretical guidance for overcoming genetic barriers to hybrid necrosis in wheat hybridization breeding, and promoting the efficient aggregation of superior traits in the breeding of new wheat varieties, which is of great significance. Attached Figure Description

[0033] Figure 1 This is a map-based clone of the wheat hybrid necrosis gene Ne1.

[0034] Figure 2 The Ne1 gene locus was analyzed using BSR-Seq.

[0035] Figure 3Genomic collinearity analysis of the Ne1 locus of the wheat hybrid necrosis gene.

[0036] Figure 4 To verify the function of the Ne1 gene in mutants.

[0037] Figure 5 To verify the function of the Ne1 gene through transgenic analysis. Detailed Implementation

[0038] The following embodiments are provided to better understand the present invention, but are not intended to limit the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] Table 1 shows the primer sequence list.

[0042]

[0043]

[0044] Example 1: Cloning and Functional Verification of the Ne1 Gene

[0045] I. Phenotypic Identification and Genetic Analysis of Leaf Necrosis

[0046] The common wheat leaf necrosis line M114 (Ne1Ne1Ne2Ne2) (described in the following literature: Yan, et al. High-temperature wheat leaf rust resistance gene Lr13 exhibits pleiotropic effects on hybrid necrosis. Molecular Plant, 2021, 14: 1029-1032) showed normal seedling characteristics, but necrosis phenotypes began to appear in the lower leaves at the jointing stage, starting with drying from the leaf tip and subsequently developing into drying and necrosis of the entire leaf; by the flowering stage, the flag leaf and the second leaf from the top also began to show obvious drying and necrosis phenotypes. The common wheat variety Zhoumai 22 (ne1ne1Ne2Ne2; denoted as Zhoumai22 in the figure) showed healthy growth in all cases. Genetic segregating populations were constructed by crossing the necrosis line M114 with Zhoumai 22 to perform genetic analysis of the Ne1 gene. In the field, leaf necrosis phenotypes were identified in the necrotic lines M114, Zhoumai 22, and the F1 hybrids of M114 × Zhoumai 22. The results showed that M114 exhibited a clear leaf necrosis phenotype, while Zhoumai 22 grew healthily. The F1 hybrids of M114 × Zhoumai 22 showed an intermediate type with localized partial leaf tip necrosis. Figure 1 a) Identification of 356 F2 individual plants revealed that 95 were homozygous and normal, while 261 showed necrosis / hemispheric necrosis, consistent with a 1:3 segregation ratio. 2:3 The family pedigrees showed a segregation ratio of 1:2:1 (Table 2).

[0047] Table 2 shows the genetic analysis of the Ne1 gene.

[0048]

[0049] χ 2 0.05 =3.841, df=1; χ 2 0.05 =5.991, df=2

[0050] The above results indicate that the Ne1 gene is an incompletely dominant gene in the necrotic line M114.

[0051] II. Fine mapping of the wheat hybrid necrosis gene Ne1

[0052] The Ne1 gene was located using BSR-seq technology. First, 50 homozygous normal and 50 homozygous necrotic families were selected from the F2 lineage of the M114 × Zhoumai 22 hybrid, respectively, to construct two extreme pools for normal and necrotic genes, followed by transcriptome sequencing. BSR-seq analysis located Ne1 on wheat chromosome 5BL. Figure 2Further development of molecular markers linked to the Ne1 gene yielded 13 such markers. Using the distal flanking markers XM11 and XM21, which are closely linked to the Ne1 gene, F2 plants of the M114 × Zhoumai 22 hybrid combination 7,235 were screened to obtain plants that underwent recombination in the Ne1 gene region. Field phenotypic identification was performed on the F2 segregating families of the screened recombinant plants.

[0053] The specific molecular markers shown in Table 1 were used to compare the necrotic line M114 (Ne1Ne1Ne2Ne2) and the variety Zhoumai 22 (ne1ne1Ne2Ne2). Figure 1 a) Construct a genetically segregating population through hybridization for genotyping, and perform phenotypic identification on key exchange individuals to estimate the genetic linkage distance of molecular markers to the Ne1 gene, and construct its fine genetic linkage map. Figure 1 b).

[0054] Genotyping of recombinant single plants was performed using molecular markers closely linked to the Ne1 gene. The Ne1 gene was ultimately finely mapped to a 0.07 cM genetic region between molecular markers XM14 and XM6. Molecular markers XM15, XM16, and XM17 co-segregated with the Ne1 gene. This region corresponds to a physical distance of 3.63 Mb from the Chinese spring reference genome (IWGSC RefSeq v1.0) and contains 15 high-confidence genes. Figure 1 c).

[0055] III. Genomic Collinearity Analysis of Ne1 Gene Locus

[0056] Based on the fine mapping of the Ne1 gene, collinearity analysis was performed using the corresponding physical regions of the reference genomes of *Strombus haemosum*, *Fielder*, *Strombus 10+*, wild emmer wheat, and durum wheat. This revealed a structural variation caused by a 2.79 Mb large genomic fragment insertion in the Ne1 gene mapping region of *Strombus haemosum* compared to other materials. Figure 3 Previous studies have reported the presence of the Ne1 gene in Chinese spring, but the allelic variation is relatively weak. Therefore, it is speculated that the Ne1 gene may be located at a large insertion site, with six genes from TraesCS5B01G212700 to TraesCS5B01G213200 being candidate Ne1 genes.

[0057] IV. Validation of Ne1 candidate gene function using EMS-induced regreen mutants

[0058] Approximately 10,000 seeds of the leaf necrosis line M114 were selected and treated with 0.6% ethyl methanesulfonate (EMS) mutagen before being sown in an experimental field. A total of 4,125 M2 generation materials were harvested. The harvested M2 seeds were planted in the field according to their numbering order. Leaf necrosis phenotypic identification was performed at the wheat flowering stage, and four regreening mutants with restored normal leaves were screened and harvested individually. These leaf regreening mutants were then sown in the field, and homozygous regreening mutants were obtained after phenotypic identification. Genetic background analysis of the obtained homozygous leaf regreening mutants and the leaf necrosis line M114 was performed using wheat genome-wide SSR molecular markers, confirming that they were regreening mutants of the necrosis line M114. The Ne1 candidate gene in the regreening mutants was amplified using specific primers, and the candidate gene base mutation sites were detected by comparison with the Ne1 candidate gene sequence in the leaf necrosis line M114. The results showed four independent mutants (…). Figure 4 (a is the EMS regreen mutant of the premature aging line M114, b is a schematic diagram of the base mutation in the Ne1 gene) There are 4 missense mutations in the TraesCS5B01G212800 gene. No mutations were found in other genes in the localization region. Therefore, the TraesCS5B01G212800 gene can be listed as a candidate gene for Ne1 for further analysis.

[0059] The Ne1 gene was cloned from the leaf necrosis line M114. Figure 1 d) The nucleotide sequence of its genome is SEQ ID No. 1, positions 2407-10563, and the cDNA sequence is shown in SEQ ID No. 2. The protein Ne1 encoded by this gene has the amino acid sequence shown in SEQ ID No. 3.

[0060] V. Transgenic verification of the function of the Ne1 candidate gene

[0061] To verify the function of the Ne1 gene, the expression vector ProNe1:Ne1, driven by the Ne1 gene's own promoter, was constructed. The Ne1 gene was then introduced into the transgenic line OE-T1-1-1 containing the Ne2 gene via Agrobacterium-mediated genetic transformation, as detailed below:

[0062] 1. Obtaining the Ne1 genome sequence

[0063] First, genomic DNA was extracted from leaves of the leaf necrosis line M114. Four Ne1 gene fragments were obtained by segmental amplification using specific primers Gne1-110, Gne1-27, Gne1-28, and Gne1-180 (Table 1). The four Ne1 gene fragments were then assembled using the pEASY-UniSeamless Cloning and Assembly Kit (Beijing TransGen Biotech Co., Ltd., CU101-01) to obtain a 13,118 bp Ne1 genome sequence (denoted as the Ne1 gene product). Its nucleotide sequence is SEQ ID No. 1, containing an 8,157 bp gene region (SEQ ID No. 1, positions 2407-10,563), a 2,406 bp upstream promoter region (SEQ ID No. 1, positions 1-2046), and a 2,556 bp downstream gene regulatory region (SEQ ID No. 1, positions 10564-13,118).

[0064] 2. Construction of Ne1 transgenic vector

[0065] The wheat expression vector pCAMBIA1300 plasmid was double-digested with restriction endonucleases BamH I and Hind III, and approximately 14,000 bp of linearized vector backbone was recovered, denoted as pCAMBIA1300 linearized vector backbone.

[0066] The 13,118 bp Ne1 gene product obtained in step 1 was homologously recombined with the pCAMBIA1300 linearized vector backbone using the pEASY-Uni Seamless Cloning and Assembly Kit (Beijing TransGen Biotech Co., Ltd., CU101-01) homologous recombination kit to obtain the recombinant plasmid ProNe1:Ne1.

[0067] Sanger sequencing further verified the integrity and correctness of the ProNe1:Ne1 vector, which can be used in the next step of the experiment.

[0068] Sequencing results showed that the recombinant plasmid ProNe1:Ne1 was obtained by inserting the Ne1 gene product shown in SEQ ID No.1 between the BamH I and Hind III sites of the pCAMBIA1300 vector, and it expressed the Ne1 gene.

[0069] The wheat expression vector pCAMBIA1300 is described in the following literature: Li et al., A CNL protein in wild emmer wheat confers powdery mildew resistance. New Phytologist, 2020, 228:1027-1037. The name in the literature is pCambia1300. The above biological material can be obtained from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained biological material is only used to repeat the experiments of this invention and should not be used for other purposes.

[0070] 3. Obtaining Ne1 transgenic plants

[0071] 1) Obtaining Ne1 transgenic plants

[0072] The recombinant plasmid ProNe1:Ne1 obtained in step 2 above was introduced into Agrobacterium EHA105 and Agrobacterium-mediated genetic transformation was performed to obtain the transgenic line OE-T1-1-1 of the Ne2 gene, resulting in the T0 generation Ne1 transgenic line.

[0073] The above-mentioned transgenic method is referenced in: Ishida Y, Tsunashima M, Hiei Y, Komari T. Wheat (Triticum aestivum L.) transformation using immature embryos. In: Wang K, ed. Agrobacterium protocols. Methods in Molecular Biology, vol. 1223. New York, NY, USA: Springer, 2015, 189.

[0074] The transgenic line OE-T1-1-1 of the Ne2 gene recipient material described above is described in the following literature: "Yan et al., High-temperature wheat leaf rust resistance gene Lr13 exhibits pleiotropic effects on hybrid necrosis. Molecular Plant, 2021, 14:1029-1032". The Ne2 gene transgenic line OE-T1-1-1 was created by stably transforming the Ne2 gene (gene number: MT992315.1; submission date: February 2, 2021) into the wheat variety Fielder using Agrobacterium-mediated genetic transformation. Its main agronomic traits are not significantly different from the transgenic recipient Fielder, but it exhibits high resistance to wheat leaf rust. The public can obtain the above-mentioned biological material from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained biological material is only for repeating the experiments of this invention and should not be used for other purposes.

[0075] 2) Molecular identification of Ne1 transgenic plants

[0076] The T0 generation Ne1 transgenic lines obtained in 1) above were molecularly identified using the following method:

[0077] Genomic DNA was extracted from the T0 generation Ne1 transgenic line to be tested. Using the genomic DNA as a template, PCR amplification was performed using the specific primer Ne1HB-jc. The recombinant expression plasmid ProNe1:Ne1 was used as a positive control, and the genomic DNA from the transgenic line OE-T1-1-1 of the Ne2 gene was used as a negative control. The expected fragment size of the amplified product was approximately 490 bp. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min. The PCR amplification products were detected by 1% agarose gel electrophoresis, photographed under UV light, and the results were recorded.

[0078] PCR testing revealed that plants with a 490bp bp molecule were positive, while those without a 490bp molecule were negative. A total of four T0 generation Ne1-positive plants were obtained and named ComNe1#1-ComNe1#4.

[0079] 4. Obtaining Ne1 transgenic plants

[0080] The four T0 generation Ne1-positive plants (ComNe1#1-ComNe1#4) obtained were self-crossed to obtain T1 generation Ne1-transgenic lines. Fifteen individual plants from each transgenic line were selected for genotypic and phenotypic identification. Details are as follows:

[0081] The T1 generation Ne1 transgenic lines were sown in the plant cultivation greenhouse of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, under a 16-hour light-8-hour dark cycle and a growth temperature of 22°C. At the two-leaf-one-heart stage of wheat, individual transgenic plants were labeled and their leaves were used to extract genomic DNA for genotyping. The detection method was consistent with the molecular identification method for the T0 generation Ne1 transgenic lines. All results yielded a 490bp fragment, confirming that all T1 generation Ne1 transgenic lines were positive T1 generation Ne1 transgenic lines. T1 generation Ne1 transgenic lines that did not yield a 490bp fragment were negative T1 generation Ne1 transgenic lines.

[0082] Leaf necrosis phenotype identification was conducted when the above-mentioned T1 generation Ne1 transgenic lines reached the grain-filling stage. Figure 5 OENE2 represents the transgenic line OE-T1-1-1 of the Ne2 gene, which is the transgenic recipient material. If half of the leaf area shows obvious drying and necrosis, it is considered to have necrosis.

[0083] The results are shown in Table 3, indicating that all T1 generation positive Ne1 transgenic lines showed leaf necrosis during the grain-filling period, while the leaves of negative T1 generation Ne1 transgenic lines and the transgenic line OE-T1-1-1 with the Ne2 gene showed normal results.

[0084] Table 3 shows the genotype and phenotypic identification results of Ne1 transgenic T1 families.

[0085]

[0086] In the table above, leaf necrosis corresponds to the T1 generation positive-transformed Ne1 strain, and normal leaf necrosis corresponds to the negative T1 generation positive-transformed Ne1 strain.

[0087] The results above demonstrate that the Ne1 gene does indeed have the function of causing hybridization necrosis together with the Ne2 gene.

[0088] Example 2: Development and application of functional markers for the wheat hybrid necrosis gene Ne1

[0089] I. Development of Functional Markers for the Wheat Hybrid Necrosis Gene Ne1

[0090] 1. Development of functional markers for the wheat hybrid necrosis gene Ne1

[0091] To effectively detect the Ne1 gene during wheat breeding, a functional marker specifically amplified, Ne1-FM, was developed based on the Ne1 gene sequence (Table 1).

[0092] Genomic DNA from leaves of the necrotic lines M114 and Zhoumai 22 was used as templates and Ne1-FM was used as primers for amplification. The amplification program was as follows:

[0093] PCR reaction system (10 μL): 2 μL of genomic DNA from wheat leaves (25 ng / μL), 5 μL of 2×PCR Mix, 1 μL of aqueous solution of one Ne1-FM primer (concentration of 10 μmol / L), 1 μL of aqueous solution of the other Ne1-FM primer (concentration of 10 μmol / L), and 1 μL of ddH2O, for a total of 10 μL.

[0094] PCR reaction conditions: 94℃ for 3 min; 94℃ for 15 s, 58℃ for 15 s, 72℃ for 15 s, 35 cycles; 72℃ for 10 min.

[0095] The amplification product of M114 showed a band size of 222bp after 1% agarose gel electrophoresis. Sequencing of the amplification product revealed that it was completely identical to the Ne1 gene sequence, while Zhoumai 22, which does not contain the Ne1 gene, showed no amplification band.

[0096] The above results indicate that Ne1-FM can specifically amplify the Ne1 gene fragment from wheat materials containing the Ne1 gene, and it can be used to detect whether the wheat to be tested contains the Ne1 gene. The specific method is as follows:

[0097] Genomic DNA was extracted from the wheat tissue (e.g., leaves) to be tested and used as a template. Ne1-FM was used as a primer for amplification. If an amplification product (222bp in size) was found, the wheat to be tested contained the Ne1 gene. If no amplification product was found, the wheat to be tested did not contain the Ne1 gene.

[0098] II. Application of the functional marker of the wheat hybrid necrosis gene Ne1 in identifying whether the offspring of the leaf necrosis line M114 (Ne1Ne1Ne2Ne2) and the common wheat variety Zhoumai 22 (ne1ne1Ne2Ne2) are necrotic.

[0099] Fifty F2 segregating populations (F2 individual plants) obtained from the cross between leaf necrosis line M114 (Ne1Ne1Ne2Ne2) and common wheat variety Zhoumai 22 (ne1ne1Ne2Ne2) were subjected to leaf necrosis phenotype statistics when they reached the grain-filling stage. The results are shown in Table 4.

[0100] Simultaneously, the 50 lines obtained from the F2 segregating population were identified using the following molecular markers:

[0101] Genomic DNA was extracted from wheat leaves as a template and amplified using Ne1-FM primers. If the amplification product contained a 222bp product (amplification was observed), the wheat sample contained the Ne1 gene. If no amplification product was observed, the wheat sample did not contain the Ne1 gene.

[0102] Using the functional marker Ne1-FM, the results of the detection of 25 necrotic materials and 25 normal materials in the F2 segregating population constructed by crossing the necrotic line M114 (Ne1Ne1Ne2Ne2) and the common wheat variety Zhoumai 22 (ne1ne1Ne2Ne2) were verified. It was found that the functional marker Ne1-FM detection results co-segregated with the phenotype. That is, the materials that amplified a 222bp band all showed necrosis, while the materials that did not amplify a band all showed normality (Table 4).

[0103] Table 4 shows the results of the functional marker Ne1-FM detection in the M114×Zhoumai 22F3 population.

[0104]

[0105]

[0106] In Table 4, "amplified" means that a target band of 222 bp can be amplified; "no amplified" means that no band is amplified.

[0107] Therefore, the functional marker Ne1-FM can be used to detect whether the hybridization necrosis gene Ne1 exists in wheat varieties. If there is an amplification product (222bp), then Ne1 is present; if there is no amplification product, then Ne1 is not present.

Claims

1. A protein comprising the amino acid sequence shown in SEQ ID No.

3.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is any one of the following: B1) The DNA molecule shown in SEQ ID No. 2; B2) The DNA molecule shown in SEQ ID No. 1 or positions 2407-10,563 of SEQ ID No.

1.

4. An expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule described in claim 2 or 3.

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