Wheat hybrid necrosis gene Ne1 cloning and functional marker development

Abstract

The invention discloses wheat hybrid necrosis gene Ne1 cloning and functional marker development. The invention provides a protein which is composed of an amino acid sequence as shown in SEQ ID No. 3. The invention also provides a nucleic acid molecule for coding the protein, which is named as Ne1 gene and is a DNA molecule as shown in SEQ ID No.2, or a DNA molecule as shown in SEQ ID No.1 or 2407th to 10th and 563rd sites as shown in SEQ ID No.1, or a DNA molecule as shown in SEQ ID No.2 or SEQ ID No.1. The invention provides gene localization, map-based cloning, mutant and transgene function verification and functional marker development of the wheat hybrid necrosis gene Ne1. The cloning of the wheat hybrid necrosis gene Ne1 can completely uncover the miracle veil formed by wheat hybrid necrosis, can provide theoretical guidance for overcoming the genetic disorder of hybrid necrosis in the wheat crossbreeding process, promotes efficient polymerization of excellent characters to cultivate new wheat varieties, and has important significance.

Description

Technical Field

[0001] The present invention belongs to the fields of crop molecular biology and molecular breeding, and particularly relates to the cloning of the wheat hybrid necrosis gene Ne1 and the development of functional markers. Background Art

[0002] Wheat is an important food crop, and about one-third of the global population takes wheat as the staple food. Hybrid breeding is currently the most common and effective breeding method for cultivating new wheat varieties. However, during the process of wheat hybrid breeding, the phenomenon of hybrid necrosis often occurs in the hybrid offspring, which greatly limits the free aggregation of excellent traits of the parents (Caldwell et al, 1943; Hermsen 1963a), and seriously hinders the genetic improvement and new variety breeding of wheat.

[0003] The hybrid necrosis of wheat is jointly controlled by a pair of complementary genes Ne1 and Ne2 (Chu et al, 2006), and when they are aggregated together through hybridization, it will cause hybrid necrosis. Although the phenomenon of wheat hybrid necrosis has been discovered for 100 years (Sax 1921), its molecular mechanism is still not very clear, making it a hot and difficult point in domestic and foreign research. Recently, multiple research teams at home and abroad have reported the cloning of one of the hybrid necrosis genes Ne2, which encodes a CC-NBS-LRR type disease-resistant protein, and proved that it is the same gene as the wheat high-temperature leaf rust resistance gene Lr13 (Yan et al, 2021; Hewitt et al, 2021; Si et al, 2021b).

[0004] Nishikawa et al. (1974) mapped the Ne1 gene to 9.4±1.5 cM from the centromere on 5BL using common wheat telosome materials (Nishikawa et al, 1974). Chu et al. (2006) constructed a genetic linkage map of Ne1 and Ne2 using SSR molecular markers. The genetic distances between Ne1 and Ne2 and their closest SSR markers Xbarc74 and Xbarc55 were 2.0 cM and 3.2 cM, respectively (Chu et al, 2006). Recently, progress has also been made in the fine mapping of the Ne1 gene. Li et al. (2021) mapped the Ne1 gene to 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) mapped the Ne1 gene between molecular markers 5B-383 and SN-2142, corresponding to a physical distance of 4.06 Mb in Chinese Spring (Si et al, 2021). Zhang et al. (2022) reported the fine mapping of the Ne1 gene to 0.5 cM between molecular markers Nwu_5B_4137 and Nwu_5B_5114 (Zhang et al, 2022).

[0005] However, the Ne1 gene has not been cloned yet. Cloning the Ne1 gene and using the unique genetic system of Ne1-Ne2 interaction to analyze the molecular mechanism of hybrid necrosis formation will completely uncover the mystery of wheat hybrid necrosis. At the same time, it can provide theoretical guidance for overcoming the genetic obstacle of hybrid necrosis in the process of wheat cross-breeding, and can also lay a theoretical foundation for the precise molecular design of disease resistance using the NLR disease-resistant protein encoded by Ne2, which is of great significance for cultivating high-yield and disease-resistant wheat varieties. Summary of the Invention

[0006] The object of the present invention is to provide the cloning of the wheat hybrid necrosis gene Ne1 and the development of functional markers.

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

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

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

[0010] A3) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID No. 3 and having the same function;

[0011] A4) A protein having a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the amino acid sequence defined in any of A1)-A3) and having the same function.

[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 described above is named gene Ne1, located on wheat chromosome 5BL, and is any of the following:

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

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

[0016] B3) A DNA molecule having a homology of more than 98% with the DNA sequence defined in B1) or B2) and encoding the same functional protein;

[0017] B4) A DNA molecule that hybridizes with the DNA sequence defined in B1) or B2) under stringent conditions and encodes the same functional protein;

[0018] B5) A DNA molecule having a homology of more than 90% with the DNA sequence defined in B1) or B2) and encoding the same functional protein.

[0019] In a third aspect, the present invention provides an expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in the second aspect.

[0020] In a fourth aspect, the present invention provides the application 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) Cultivating a plant with a necrosis phenotype, wherein the plant contains the Ne2 gene;

[0022] C2) Making a plant containing the Ne2 gene have a necrosis phenotype;

[0023] C3) Acting together with the Ne2 gene to cause plant necrosis.

[0024] In a fifth aspect, the present invention provides the preparation of a transgenic plant with a necrosis phenotype, including 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 a primer pair for amplifying the full length or a part of the nucleic acid molecule described in the second aspect, which is composed of the single-stranded DNA molecule shown in SEQ ID No. 4 and the single-stranded DNA molecule shown in SEQ ID No. 5.

[0026] In a seventh aspect, the present invention provides a PCR reagent or kit containing the primer pair described in the sixth aspect.

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

[0028] In the above text, the plant variety may be the hybrid offspring of plants containing the Ne1 and Ne2 genes. Further specifically, it is the hybrid offspring of the necrosis line M114. In the examples of the present invention, it is the hybrid offspring of the necrosis line M114 and the common wheat variety Zhoumai 22.

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

[0030] The present invention uses EMS mutagenesis to create a re-greening mutant of the necrosis line M114, and its phenotype is as shown in ( Figure 4 ).

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

[0032] The present invention provides gene mapping, map-based cloning, mutant and transgenic functional verification, and functional marker development of the wheat hybrid necrosis gene Ne1. The cloning of the wheat hybrid necrosis gene Ne1 will completely uncover the mystery of the formation of wheat hybrid necrosis, provide theoretical guidance for overcoming the genetic obstacle of hybrid necrosis in the process of wheat hybrid breeding, promote the efficient polymerization of excellent traits to cultivate new wheat varieties, and is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 For map-based cloning of the wheat hybrid necrosis gene Ne1.

[0034] Figure 2 For BSR-Seq analysis of the Ne1 gene locus.

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

[0036] Figure 4 To verify the function of the Ne1 gene by mutant experience.

[0037] Figure 5 To verify the function of the Ne1 gene by transgenic technology. Specific implementation mode

[0038] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

[0039] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0040] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0041] Table 1 is the primer sequence table

[0042]

[0043]

[0044] Example 1: Cloning and functional verification of the Ne1 gene

[0045] I. Identification of leaf necrosis phenotype and genetic analysis

[0046] The common wheat leaf necrosis line M114 (Ne1Ne1Ne2Ne2) (recorded 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) shows normal growth at the seedling stage, but at the jointing stage, necrosis phenotypes begin to appear in the lower leaves, starting from the leaf tip and then developing into dry necrosis of the whole leaf; at the wheat flowering stage, obvious dry necrosis phenotypes also begin to appear in the flag leaf and the second leaf from the top; the common wheat variety Zhoumai 22 (ne1ne1Ne2Ne2; denoted as Zhoumai22 in the figure) shows healthy growth. The genetic analysis of the Ne1 gene was carried out using the necrosis line M114 and Zhoumai 22 to construct a genetic segregation population. In the field, the necrosis line M114, Zhoumai 22, and the F of the M114×Zhoumai 22 combination 1The hybrid was identified for leaf necrosis phenotype. It was found that the necrosis line M114 showed an obvious leaf necrosis phenotype, while Zhoumai 22 grew healthily. The F of the M114 × Zhoumai 22 combination 1 hybrids showed an intermediate phenotype of local semi-necrosis at the leaf tip ( Figure 1 a). Among the 356 F individual plants identified, 95 showed homozygous normality, and 261 showed necrosis / semi-necrosis, conforming to the segregation ratio of 1:3. The F 2 families conformed to the segregation ratio of 1:2:1 (Table 2). 2:3

[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 necrosis line M114.

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

[0052] The BSR-seq technology was used to map the Ne1 gene. First, 50 homozygous normal and 50 homozygous necrosis families were selected from the F families of the M114 × Zhoumai 22 combination to construct two extreme bulk pools of normal and necrosis respectively for transcriptome sequencing. BSR-seq analysis mapped Ne1 on wheat chromosome 5BL ( 2 ). Further, molecular markers linked to the Ne1 gene were developed, and a total of 13 molecular markers linked to the Ne1 gene were obtained. The relatively distant flanking molecular markers XM11 and XM21 closely linked to the Ne1 gene were used to screen 7,235 F individual plants of the M114 × Zhoumai 22 combination, Figure 2 ) and single plants with recombination in the Ne1 gene region were obtained. The F 2 segregation families of the screened recombinant single plants were subjected to field phenotype identification. 2

[0053] Using the specific molecular markers shown in Table 1, the necrosis line M114 (Ne1Ne1Ne2Ne2) and the cultivar Zhoumai 22 (ne1ne1Ne2Ne2) ( Figure 1 a) were hybridized to construct a genetic segregation population for genotype identification, and the key crossover single plants were phenotypically identified to estimate the genetic linkage distance of the molecular markers from the Ne1 gene, and a fine genetic linkage map was constructed ( Figure 1 b).

[0054] ​​The recombinant individuals were genotyped using molecular markers closely linked to the Ne1 gene within the mapping interval. Finally, the Ne1 gene was finely mapped to a genetic interval of 0.07 cM between molecular markers XM14 and XM6. Among them, molecular markers XM15, XM16, and XM17 were co-segregated with the Ne1 gene. The physical distance of this interval corresponding to the Chinese Spring reference genome (IWGSC RefSeq v1.0) was 3.63 Mb, containing 15 high-confidence genes( Figure 1 c).

[0055] III. Genome collinearity analysis of the Ne1 gene locus

[0056] Based on the fine mapping of the Ne1 gene, collinearity analysis was performed using the corresponding physical intervals of the reference genomes of Chinese Spring, Fielder, common wheat 10+ genomes, wild emmer wheat, and durum wheat. It was found that compared with other materials, there was a structural variation caused by a 2.79-Mb genomic large fragment insertion in the Ne1 gene mapping interval of Chinese Spring( Figure 3 ). Previous studies reported that Chinese Spring contains the Ne1 gene, but the allelic variation belongs to a weaker type. Therefore, it is speculated that the Ne1 gene may be located on the large fragment insertion, and 6 genes from TraesCS5B01G212700 to TraesCS5B01G213200 are candidate genes for Ne1.

[0057] IV. Verification of the function of Ne1 candidate genes using EMS-induced green-revertant mutants

[0058] Approximately 10,000 seeds of the leaf necrosis line M114 were selected, treated with 0.6% ethyl methanesulfonate (EMS) mutagen, and then sown in the experimental field. Finally, a total of 4,125 M 2 generation materials were harvested. The M 2 harvested seeds were planted in the field, and the leaf necrosis phenotype was identified at the wheat flowering stage. A total of 4 green-revertant mutants with normal leaves were screened and harvested individually. Then, the leaf green-revertant mutants were sown in the field, and homozygous green-revertant mutants were obtained through phenotype identification. Wheat whole-genome SSR molecular markers were used to detect the genetic background of the obtained homozygous leaf green-revertant mutants and the leaf necrosis line M114, and it was determined that they were green-revertant mutants of the necrosis line M114. Specific primers were used to amplify the Ne1 candidate genes in the green-revertant mutants, and the base mutation sites of the candidate genes were detected by comparing with the Ne1 candidate gene sequences in the leaf necrosis line M114. The results showed that there were 4 independent mutants( Figure 4, a is an EMS re-greening mutant of the early senescence line M114, and b is a schematic diagram of the base mutation of the Ne1 gene. There are 4 missense mutations in the TraesCS5B01G212800 gene, and no mutations are found in other genes in the mapped interval. Therefore, the TraesCS5B01G212800 gene can be listed as a Ne1 candidate gene 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 from positions 2407 to 10563 of SEQ ID No.1, and the cDNA sequence is as shown in SEQ ID No.2. The protein Ne1 encoded by this gene has an amino acid sequence as shown in SEQ ID No.3.

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

[0061] To verify the gene function of Ne1, an expression vector ProNe1:Ne1 driven by the Ne1 gene's own promoter was constructed, and the Ne1 gene was introduced into the transgenic line OE-T of the Ne2 gene through Agrobacterium-mediated genetic transformation 1 -1-1, specifically as follows:

[0062] 1. Obtaining the Ne1 genomic sequence

[0063] First, genomic DNA was extracted from the leaves of the leaf necrosis line M114, and 4 Ne1 gene fragment products were obtained by segmental amplification using the specific primers Gne1-110, Gne1-27, Gne1-28, and Gne1-180 in Table 1. The pEASY-UniSeamless Cloning and Assembly Kit (TransGen Biotech Co., Ltd., Beijing, CU101-01) homologous recombination kit was used to splice the 4 Ne1 gene fragment products to obtain a 13,118 bp Ne1 genomic sequence (denoted as the Ne1 gene product), whose nucleotide sequence is SEQ ID No.1, which contains an 8,157 bp gene region (positions 2407 to 10,563 of SEQ ID No.1), a 2,406 bp upstream promoter region (positions 1 to 2046 of SEQ ID No.1), and a 2,556 bp downstream gene regulatory region (positions 10564 to 13,118 of SEQ ID No.1).

[0064] 2. Construction of the Ne1 transgenic vector

[0065] The wheat expression vector pCAMBIA1300 plasmid was digested with the restriction enzymes BamH I and HindIII, and the linearized vector backbone of approximately 14,000 bp was recovered and denoted as the pCAMBIA1300 linearized vector backbone.

[0066] Using the pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech Co., Ltd., Beijing, CU101-01) homologous recombination kit, the 13,118-bp Ne1 gene product obtained above and the pCAMBIA1300 linearized vector backbone were subjected to homologous recombination to obtain the recombinant plasmid ProNe1:Ne1.

[0067] The ProNe1:Ne1 vector was further verified to be complete and correct by Sanger sequencing and could be applied to the next experiment.

[0068] The sequencing results showed that the recombinant plasmid ProNe1:Ne1 was a vector obtained by inserting the Ne1 gene product shown in SEQ ID No.1 between the BamH I and HindIII 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, and its name in the literature is pCambia1300. The public can obtain the above biological material from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained above biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0070] 3. Obtaining Ne1 transgenic plants

[0071] 1) Obtaining transgenic Ne1 plants

[0072] The recombinant plasmid ProNe1:Ne1 obtained above was introduced into Agrobacterium tumefaciens EHA105, and Agrobacterium-mediated genetic transformation was performed on the transgenic line OE-T of the Ne2 gene 1 -1-1 to obtain the T 0 generation transgenic Ne1 lines.

[0073] The above transgenic method reference: 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-T of the above transgenic receptor material Ne2 gene 1 -1-1 is recorded 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 transgenic line OE-T of Ne2 gene 1 -1-1 was stably transformed into the wheat variety Fielder through Agrobacterium-mediated genetic transformation experiments with the Ne2 gene (gene number: MT992315.1; submission date: February 2, 2021). Its main agronomic traits have no obvious differences from those of the transgenic receptor Fielder, but it shows high resistance to wheat leaf rust. The public can obtain the above biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained above biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0075] 2) Molecular identification of Ne1 transgenic plants

[0076] The T 0 generation of Ne1 transgenic lines obtained in 1) above were subjected to molecular identification according to the following method:

[0077] Extract the genomic DNA of the T 0 generation of Ne1 transgenic lines to be tested. Using the genomic DNA as a template, perform PCR amplification with the specific primer Ne1HB-jc, using the recombinant expression plasmid ProNe1:Ne1 as a positive control, and the transgenic line OE-T of Ne2 gene 1The genome of -1-1 was used as a negative control for PCR amplification. The expected size of the amplified product fragment was approximately 490 bp. The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 30 s, for 35 cycles; extension at 72°C for 10 min. The PCR amplification products were detected by 1% agarose gel electrophoresis, photographed under ultraviolet light, and the results were recorded.

[0078] By PCR detection, plants with a 490-bp band were positive plants, and plants without a 490-bp band were negative plants. A total of 4 T 0 -generation transgenic plants overexpressing Ne1 were obtained and named ComNe1#1 - ComNe1#4 respectively.

[0079] 4. Obtaining of Ne1 transgenic plants

[0080] The 4 T 0 -generation transgenic plants overexpressing Ne1, ComNe1#1 - ComNe1#4, obtained above were self-crossed to obtain T 1 -generation transgenic lines overexpressing Ne1. For each transgenic line, 15 individual plants were selected for genotype and phenotype identification. The details are as follows:

[0081] T 1 The T 0 -generation transgenic lines overexpressing Ne1 were sown in the plant culture greenhouse of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, with a 16-h light and 8-h dark cycle and a growth temperature of 22°C. At the two-leaf and one-heart stage of wheat, labels were attached to distinguish individual plants, and leaves of transgenic plants were taken to extract genomic DNA for genotype detection. The detection method was the same as that for the molecular identification of the T 1 -generation transgenic lines overexpressing Ne1. The results all showed a 490-bp fragment, proving that the T 1 -generation transgenic lines overexpressing Ne1 were all positive T 1 -generation transgenic lines overexpressing Ne1, and T 1 -generation transgenic lines overexpressing Ne1 without a 490-bp band were negative T

[0082] When the above-mentioned T 1 -generation transgenic lines overexpressing Ne1 grew to the filling stage, leaf necrosis phenotype identification was carried out ( Figure 5 , where OENE2 represents the transgenic line OE-T 1 -1-1 of the Ne2 gene of the transgenic receptor material). If obvious dry necrosis occurred in half of the leaf area, it was considered that necrosis occurred.

[0083] The results are shown in Table 3, indicating that all T 1 -generation positive transgenic lines overexpressing Ne1 showed leaf necrosis at the filling stage, while negative T 1 -generation transgenic lines overexpressing Ne1 and the transgenic line OE-T 1The leaves of -1-1 all showed normal conditions.

[0084] Table 3 shows the genotype and phenotype identification results of the Ne1 transgenic T 1 family lines

[0085]

[0086] In the above table, the T 1 generation positive Ne1 transgenic lines corresponded to leaf necrosis, and the negative T 1 generation Ne1 transgenic lines corresponded to normal conditions.

[0087] The above results indicate that the Ne1 gene indeed has the function of causing hybrid necrosis together with the Ne2 gene.

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

[0089] I. Development of a functional marker for the wheat hybrid necrosis gene Ne1

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

[0091] In order to effectively detect the Ne1 gene during wheat breeding, a specific amplification functional marker Ne1-FM (Table 1) was developed based on the Ne1 gene sequence.

[0092] Using the genomic DNA of the necrotic line M114 and Zhoumai 22 leaves as templates, and Ne1-FM as primers for amplification. The amplification program was as follows:

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

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

[0095] Among them, the amplification product of M114 was detected by 1% agarose gel electrophoresis, and the band size was 222 bp. After sequencing the amplification product, it was found that it was completely consistent with the Ne1 gene sequence, while Zhoumai 22 without the Ne1 gene had 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] Extract the genomic DNA of the wheat tissue to be tested (such as leaves) as a template, and use Ne1-FM as a primer for amplification. If there is an amplification product (a product with a size of 222 bp), then the wheat to be tested contains the Ne1 gene. If there is no amplification product, then the wheat to be tested does not contain the Ne1 gene.

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

[0099] Hybridize the leaf necrosis line M114 (Ne1Ne1Ne2Ne2) and the common wheat variety Zhoumai 22 (ne1ne1Ne2Ne2) to construct the F 2 When 50 lines (F2 individual plants) obtained from the segregating population grow to the filling stage, the leaf necrosis phenotypes are counted. The results are shown in Table 4.

[0100] At the same time, the 50 lines obtained from the F 2 segregating population are subjected to the following molecular marker identification:

[0101] Extract the genomic DNA of the wheat leaves to be tested as a template, and use Ne1-FM as a primer for amplification. If the amplification product contains a 222-bp product (there is amplification), then the wheat to be tested contains the Ne1 gene. If there is no amplification product, then the wheat to be tested does not contain the Ne1 gene.

[0102] Using the functional marker Ne1-FM to detect 25 necrotic materials and 25 normal materials in the F 2 segregating population constructed by crossing the necrosis line M114 (Ne1Ne1Ne2Ne2) and the common wheat variety Zhoumai 22 (ne1ne1Ne2Ne2), it is found that the detection results of the functional marker Ne1-FM are co-segregated with the phenotypes, that is, the materials with an amplified 222-bp band all show necrosis, while the materials without an amplified band all show normal (Table 4).

[0103] Table 4 shows the detection of the M114×Zhoumai 22 F 3 population

[0104]

[0105]

[0106] In Table 4, "with amplification" means that a target band of 222 bp can be amplified; "without amplification" means that no band is amplified.

[0107] Therefore, the functional marker Ne1-FM can be used to detect whether the hybrid necrosis gene Ne1 exists in wheat varieties. If an amplification product (222 bp) is obtained, it contains Ne1; if no amplification product is obtained, it does not contain Ne1.

Claims

1. A protein, which is a protein shown in any one of the following A1)-A4): A1) a protein consisting of the amino acid sequence shown in SEQ ID No. 3; A2) a fusion protein obtained by connecting a tag to the N-terminus and / or the C-terminus of the protein shown in SEQ ID No. 3; A3) a protein having the same function as the amino acid sequence shown in SEQ ID No. 3 after one or more amino acid residues are replaced and / or deleted and / or added; A4) A protein having 99% or more, 95% or more, 90% or more, 85% or more or 80% homology with the amino acid sequence defined in any one of A1) to A3) and having the same function.

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 of the following: B1) DNA molecule shown in SEQ ID No.2; B2) the DNA molecule shown in SEQ ID No. 1 or positions 2407 to 10,563 of SEQ ID No. 1; B3) a DNA molecule having more than 98% homology with the DNA sequence defined in B1) or B2) and encoding a protein with the same function; B4) a DNA molecule that hybridizes with the DNA sequence defined in B1) or B2) under stringent conditions and encodes a protein with the same function; B5) A DNA molecule having more than 90% homology with the DNA sequence defined in B1) or B2) and encoding a protein with the same function.

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

5. Use of the protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the expression cassette, recombinant vector or recombinant microorganism according to claim 4 in any of the following: C1) cultivating plants having a necrotic phenotype, wherein the plants contain the Ne2 gene; C2) in making plants containing the Ne2 gene have a necrotic phenotype; C3) works together with Ne2 gene to cause plant necrosis.

6. A method for preparing a transgenic plant having a necrotic phenotype, comprising the following steps: introducing the nucleic acid molecule according to claim 2 or 3 into a target plant containing the Ne2 gene to obtain a transgenic plant having a necrotic phenotype.

7. A primer pair for amplifying the whole or part of the nucleic acid molecule according to claim 2 or 3, which consists of a single-stranded DNA molecule shown in SEQ ID No. 4 and a single-stranded DNA molecule shown in SEQ ID No.

5.

8. A PCR reagent or kit containing the primer pair according to claim 7.

9. Use of the primer pair according to claim 7 or the PCR reagent or kit according to claim 8 in identifying whether a wheat variety contains the nucleic acid molecule according to claim 2 or 3.

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