Rye resistance genes to stripe rust and their application

Abstract

The present application provides an anti-rust gene and application thereof, the nucleotide sequence of the anti-rust gene is shown as SEQ ID NO:2 or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the nucleotide sequence shown as SEQ ID NO:2.The anti-rust gene of the present application is overexpressed in the material tobacco 19 with high susceptibility to rust, and the transgenic positive plants show near immunity.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and relates to the stripe rust resistance gene of rye SECCE6Rv1G0452990 and its application. Background Technology

[0002] Wheat (Triticum aestivum L.) is one of China's important food crops, and maintaining and increasing its yield is a crucial guarantee for national food security. Wheat stripe rust is a common disease in wheat production, caused by Puccinia striiformis Westend.f.sp.Tritici, affecting the leaves. The most obvious characteristic of stripe rust is the production of conspicuous yellow or orange spores on the leaves, generally arranged in rows along the veins. 88% of wheat-producing areas worldwide are susceptible to stripe rust, resulting in an annual wheat yield reduction of 5 million tons and economic losses of up to $1 billion (see reference: Colin, R, Wellings. Global status of stripe rust: a review of historical and current threats. Euphaitica. 2011, 179:129-141). China is one of the world's largest areas affected by stripe rust, having experienced four major epidemics since the founding of the People's Republic of China, in 1950, 1964, 1990, and 2002, resulting in a yield loss of approximately 13 billion kilograms (see reference: Wan A, Zhao Z, Chen X, et al. Wheatstripe rust epidemic and virulence of Puccinia striiformis f.sp. triticiin China in 2002. Plant Disease, 2004, 88: 896-904). Therefore, the breeding of stripe rust-resistant wheat varieties and the discovery and utilization of disease-resistant genes are of great significance to ensuring national food security.

[0003] Modern breeding focused on high yields has significantly diminished crop genetic diversity. This loss not only limits further improvements in yield and quality but also makes crops more vulnerable to global climate change and the spread of pests and diseases. Wheat's close relatives contain many superior genes and are important genetic resources for improving common wheat varieties. Therefore, introducing beneficial genes from wheat's close relatives into common cultivated wheat is one of the important and effective approaches to wheat variety improvement.

[0004] Rye (Secale cereale L.) is an annual or biennial herbaceous crop belonging to the genus Secale in the family Poaceae, and is one of the wild relatives of wheat. Rye is a diploid species with a chromosome set of RR. It possesses desirable traits lacking in commonly cultivated wheat and is an important wild germplasm resource for the genetic improvement of common wheat. Triticale is a new species artificially synthesized from species of the genera *Triticum* and *Secale* through intergeneric sexual hybridization and chromosome doubling. It combines the high yield and quality of wheat with the disease resistance, cold resistance, and high lysine content of rye, exhibiting significant hybrid vigor and suitable for use as feed, brewing, bioenergy, and food. Triticale, resulting from the hybridization of diploid rye with wheat of different ploidy levels, mainly falls into two types: hexaploid (AABBRR) and octoploid (AABBDDRR).

[0005] Currently, there are no literature reports on the research content of stripe rust resistance gene sequences and molecular markers in Rozovskaya-7 triticale. Summary of the Invention

[0006] This invention used the stripe rust physiological race CYR32-34 for inoculation and identification, and found that the two triticale parents, Rozovskaya-7 and 4100, showed near-immunity and high susceptibility, respectively. The two triticale parents were crossed to obtain F1, and self-crossing yielded the F2 population. Inoculation and identification of 200 individual plants in the F2 population revealed that 144 were resistant and 56 were highly susceptible. From the F2 population, 40 extremely resistant plants and 40 extremely susceptible plants were selected to form a resistance pool and a susceptibility pool, respectively. Total RNA was extracted from leaves and sequenced. BSR (Bulk segregant RNA sequencing) analysis revealed that the stripe rust resistance gene was located at the 6RL terminus of the triticale. Using resequencing data from the two parents, 11 co-dominant Indel markers were developed near the 6RL terminus. Fine mapping was performed using the F2 population of 1246 individual plants, further mapping the stripe rust resistance gene to a 1.25 Mb interval (corresponding to Lo7 reference genome 6R: 878.556-879.809 Mb). Subsequently, analysis of 24 genes within the interval using transcriptome data from both the resistant and susceptible rye regions revealed that 19 genes had an FPKM <1 in both regions, suggesting they might be pseudogenes. Of the remaining 5 genes, only SECCE6Rv1G0452990 showed higher expression levels in the resistant region than in the susceptible region, thus identifying SECCE6Rv1G0452990 as a candidate gene. Using the rye reference genome sequence (https: / / ftp.ebi.ac.uk / ensemblgenomes / pub / release-56 / plants / fasta / secale_cere ale / dna / ), the SECCE6Rv1G0452990 sequence from Rozovskaya-7 was cloned, and the specific molecular marker 6R2990-1 was developed. To verify the function of SECCE6Rv1G0452990, genetic transformation was performed using an overexpression vector of SECCE6Rv1G0452990 on wheat susceptible to stripe rust. The transgenic results showed that overexpression of SECCE6Rv1G0452990 could improve wheat resistance to stripe rust, indicating that SECCE6Rv1G0452990 is a stripe rust resistance gene.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] On one hand, the present invention provides a stripe rust resistance gene, characterized in that the nucleotide sequence of the stripe rust resistance gene is as shown in SEQ ID NO:2 or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO:2.

[0009] On the other hand, the present invention provides an expression vector, characterized in that the expression vector contains the stripe rust resistance gene as described above.

[0010] On the other hand, the present invention provides a host cell, characterized in that the host cell contains the stripe rust resistance gene or expression vector as described above.

[0011] On the other hand, the present invention provides a method for detecting the presence or absence of a stripe rust resistance gene in a plant to be tested, characterized in that the method includes detecting the presence of the stripe rust resistance gene or its specific molecular marker according to claim 1, preferably the sequence of the specific molecular marker is as shown in SEQ ID NO:3, and preferably using primers shown in SEQ ID NO:4 and SEQ ID NO:5 to detect the presence or absence of the sequence in SEQ ID NO:3.

[0012] On the other hand, the present invention provides a method for improving the ability of plants to resist stripe rust, the method comprising introducing a stripe rust resistance gene or expression vector or host cell as described above into the plant or the cells of the plant.

[0013] On the other hand, the present invention provides a method for cultivating plants resistant to stripe rust, the method comprising introducing a stripe rust resistance gene or expression vector or host cell as described above into the plant or the cells of the plant.

[0014] On the other hand, the present invention provides the application of the stripe rust resistance gene or expression vector or host cell as described above in improving the ability of plants to resist stripe rust or in cultivating stripe rust-resistant plants.

[0015] On the other hand, the present invention provides a method for obtaining plant cells or plants carrying a stripe rust resistance gene, characterized in that the method includes introducing the stripe rust resistance gene or expression vector or host cell as described above into the plant cells or plants.

[0016] On the other hand, the present invention provides a molecular marker for detecting the presence or absence of a stripe rust resistance gene in a plant, characterized in that the nucleotide sequence of the molecular marker is shown in SEQ ID NO:3.

[0017] In some implementations, the plant is wheat or rye.

[0018] In some embodiments, the stripe rust is caused by physiological races CYR32, CYR33 and / or CYR34.

[0019] definition

[0020] GISH: Genomic in situ hybridization, is a technique developed based on fluorescence in situ hybridization. This technique uses total genomic DNA from one species as a label probe, while total genomic DNA from another species is used as a blocking agent at an appropriate concentration. In situ hybridization then occurs on the target chromosome. Between the blocking DNA and the label probe, the blocking DNA preferentially hybridizes with non-specific sequences, while the remaining specific sequences are primarily hybridized with the label probe. Finally, chromosome sets, chromosomes, or chromosome fragments from different sources are detected.

[0021] Inoculation identification: Healthy rye seedlings were cultivated in a greenhouse and inoculated with stripe rust spores by artificial sprinkling. After cultivation under moist conditions and light, the wheat plants were induced to develop the disease in order to identify the stripe rust resistance of the materials.

[0022] Near-immune and highly susceptible: Based on the degree of resistance of wheat varieties to stripe rust, the disease resistance of wheat varieties can be divided into six levels: immune, highly resistant, resistant, moderately resistant, moderately susceptible, and highly susceptible. Immune is characterized by the absence of any visible spore masses on the leaves. Near-immune is characterized by the appearance of small necrotic spots on the leaves, without the production of spore masses. Highly susceptible is characterized by large and numerous spore masses on the leaves, with no chlorosis around the spores.

[0023] Extreme disease resistance and extreme disease susceptibility: The study of disease resistance inheritance mainly employs Mendelian analysis, which involves crossing resistant and susceptible plants and analyzing the segregation of disease resistance in the offspring. In the F2 segregating population, the most resistant material is the extremely resistant material, and the most susceptible material is the extremely susceptible material.

[0024] BSR (Bulked Segregant Analysis) is a rapid method for locating genes of a target trait using pooled samples of individuals exhibiting extreme traits. In a segregating population, progeny DNA from individuals exhibiting extreme traits is pooled in equal amounts. Sequencing is then performed on both the pooled gene pool and the parents. The allele frequency (AF) of sequencing data from different libraries at specific site-specific polymorphisms (SNPs) is calculated and correlated with the target phenotype, thereby enabling the localization of genes associated with the target trait or the development of linkage molecular markers.

[0025] Codominant markers: Genetic markers that can detect both dominant and recessive alleles and distinguish between homozygous and heterozygous genotypes.

[0026] Indel markers: Insertion-deletion (InDel) refers to the insertion or deletion of nucleotide fragments of different sizes at the same locus in the genome of closely related species or different individuals of the same species. Specifically, it means that one or more bases are inserted or deleted at a certain locus in a sequence compared to another homologous sequence. InDel polymorphic molecular markers are markers used for PCR amplification based on specific primers designed for the sequences flanking the insertion / deletion site. Essentially, they are still length polymorphism markers and can be genotyped using convenient electrophoresis platforms.

[0027] Ubiquitin promoter: Ubiquitin (UBQ or Ubi) gene promoters, as constitutive promoters, show significantly high gene expression levels in most tissues and many cell types of plants, especially in monocotyledonous plants (such as maize), where they are often used to drive the overexpression of a gene.

[0028] Specific molecular markers: Specific molecular markers are developed based on functional single nucleotide polymorphisms (SNPs) or insertions and deletions (InDels) sites in gene sequences. They have advantages such as high selection efficiency, co-dominance, co-segregation with the target gene, and no influence from genetic background.

[0029] FPKM (Fragments Per Kilobase of exon model per Million fragments mapped) is a commonly used quantitative method for gene expression, used to measure the expression level of a gene (or transcript). FPKM is a formula for measuring the relative expression level of a gene. It is calculated by dividing the number of fragments mapped to the gene by the total number of sequencing reads mapped to the genome (in milliseconds) and the length of the RNA (in kilobytes).

[0030] Genome resequencing: A method for sequencing the genomes of different individuals within a species whose genome sequence is known. It is used to analyze differences between genomes of different individuals, such as identifying single nucleotide polymorphism sites, insertion / deletion sites, structural variation sites, and copy number variation sites.

[0031] 1BL / 1RS translocation lines are wheat-rye translocation chromosomes formed by translocating the short arm of rye chromosome 1R to the long arm of wheat chromosome 1B. Because the short arm of chromosome 1R carries genes for resistance to wheat leaf rust, stem rust, stripe rust, and powdery mildew, germplasm containing 1BL / 1RS has been widely used in wheat breeding worldwide. Attached Figure Description

[0032] Figure 1This study investigated stripe rust resistance identification and chromosomal composition analysis using GISH for hexaploid triticale Rozovskaya-7. Figure a shows the stripe rust identification of hexaploid triticales Rozovskaya-7 and 4100, with Mingxian169 used as a susceptible control. Figures b and c show the GISH analysis of Rozovskaya-7 and 4100, respectively, using the rye genome as a probe (green). Stripe rust inoculation results indicated that Rozovskaya-7 is immune to stripe rust. GISH results showed that both Rozovskaya-7 and 4100 are hexaploid triticales.

[0033] Figure 2 Results of stripe rust identification for hexaploid triticale Rozovskaya-7 and 4100F2 plants. F2 plants were inoculated at the one-leaf stage with a mixture of three physiological races (CYR32, CYR33, and CYR34). Phenotypic analysis was performed 14 days after inoculation. 1, 4100; 2, Rozovskaya-7; 3-5, resistant F2 plants; 6-8, susceptible F2 plants. The results indicate segregation of resistance within the F2 population, with significant differences between resistant and susceptible F2 plants.

[0034] Figure 3 The results of amplification of the SECCE6Rv1G0452990-specific molecular marker 6R2990-1 in triticale, common wheat, and transgenic seedlings. From left to right: Marker, 6 common wheat varieties (Yannong 19, Zhongguochun, Fielder, Shannong 19, Shannong 20 and Shannong 122), 3 1BL / 1RS translocation lines (Aikang 58, Kenong 199 and Nongda 399), and 18 hexaploid triticale varieties (4100, Rozovskaya-7, L20191212, PI 542533, PI 542560, PI 552974, PI 559373, PI590946, PI 610226, CIXT33, PI 511870, PI 587384, PI542563, PI 610224, PI 610227, PI628658, PI 610225, CIXT101). Tubilin primer amplification results showed that the DNA used in this invention was of reliable quality. 6R2990-1 amplification results indicated that SECCE6Rv1G0452990 was undetectable in six common wheat and three 1BL / 1RS translocation lines, but detectable in four triticale lines, and undetectable in 14 triticale lines. These results indicate that SECCE6Rv1G0452990 is not present in common wheat and 1BL / 1RS translocation lines, but is present in triticale.

[0035] Figure 4The results of stripe rust resistance in transgenic positive plants are shown. A mixture of three physiological races (CYR32, CYR33, and CYR34) was inoculated at the one-leaf-one-heart stage, and phenotypes were analyzed 14 days after inoculation. The inoculation results showed that the SECCE6Rv1G0452990 transgenic positive material was nearly immune to stripe rust, while the SECCE6Rv1G0452990 transgenic negative material was highly susceptible to stripe rust, proving that SECCE6Rv1G0452990 is a stripe rust resistance gene. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0037] Example 1: Identification of the stripe rust resistance gene in rye

[0038] The inventors used three physiological races of stripe rust, CYR32, CYR33, and CYR34 (inoculated at the stripe rust identification platform of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences), for mixed inoculation (the inoculation method refers to the powdering method in section 3.3, "Propagation and Preservation of Wheat Stripe Rust," of the national standard NY / T 1443.1-2007. In practice, the leaves only need to be covered with a mixture of white talcum powder and spores; the inoculation effect is judged by the disease incidence of the susceptible control material). The results showed that two triticale parents, Rozovskaya-7 (preserved by Han Fangpu's laboratory at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) and 4100 (provided by Professor Ni Zhongfu of China Agricultural University), exhibited near-immunity and high susceptibility to the three mixed physiological races CYR32, CYR33, and CYR34, respectively. To clone the stripe rust resistance gene in Rozovskaya-7, the two triticale parents were crossed to obtain F1, and then self-crossed to obtain the F2 population. Inoculation and identification of 200 individual plants in the F2 population revealed 144 resistant and 56 highly susceptible plants. From the F2 population, 40 extremely resistant and 40 extremely susceptible plants were selected to form a resistance pool and a susceptible pool, respectively. Total RNA was extracted from leaves and sequenced (transcriptome sequencing was performed at Annoroad Gene Technology Co., Ltd.). BSR (Bulk segregant RNA sequencing) analysis showed that the stripe rust resistance gene was located at the end of the 6RL region of rye. Eleven co-dominant Indel markers were developed near the 6RL region using genome resequencing data from two parents at a 10x sequencing depth. Fine mapping was performed using an F2 population of 1246 individual plants (the F2 progeny obtained after crossing the two parents; this was the population used for gene mapping, with a resistance:susceptibility ratio of 3:1). The stripe rust resistance gene was further mapped to a 1.25 Mb region (corresponding to Lo7 reference genome 6R: 878.556-879.809 Mb). Subsequently, analysis of 24 genes within the interval was performed using transcriptome data from both the anti-crystal and susceptible crystal regions. Nineteen genes showed FPKM < 1 in both regions, suggesting they might be pseudogenes. Of the remaining five genes, only SECCE6Rv1G0452990 showed higher expression levels in the anti-crystal region than in the susceptible crystal region (Table 2), thus identifying SECCE6Rv1G0452990 as a candidate gene.

[0039] Table 1 shows the statistical results of stripe rust identification in the Rozovskaya-7 and 4100F2 populations. A total of 200 plants were inoculated, with 144 resistant and 56 highly susceptible, basically meeting the 3:1 segregation ratio.

[0040]

[0041] Table 2 Screening of disease resistance genes

[0042]

[0043] Example 2: Detection of stripe rust resistance gene in rye

[0044] The genomic DNA of the plant materials to be tested was amplified by PCR using the molecular marker 6R2990-1 primer. All materials that could amplify 1776bp were those containing SECCE6Rv1G0452990.

[0045] Primer F for 6R2990-1: 5'-CCAGAGAGCGTGTTATGGCT-3' (SEQ ID NO:4);

[0046] Primer R for 6R2990-1: 5'-AGACGGAGATAGATTGTTCACTT-3' (SEQ ID NO:5).

[0047] The F primer is located in the promoter region of the gene, and the R primer is located at the front end of the CDS region.

[0048] Table 3 PCR System

[0049]

[0050] PCR reaction procedure: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 1 minute, run for 35 cycles; final extension at 72℃ for 10 minutes. PCR amplification products can be stored at 4℃.

[0051] PCR product electrophoresis detection system: 1.2% agarose gel electrophoresis, voltage: 120V, time: 25 minutes, buffer: 1×TAE.

[0052] Example 3 Functional identification of rye stripe rust resistance gene

[0053] An overexpression vector of SECCE6Rv1G0452990 was constructed using the Ubiqutin promoter. First, the wheat transgenic vector pWMB110 (provided by Professor Ye Xingguo of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences) was linearized using restriction endonucleases kpnI and SacI. The SECCE6Rv1G0452990 CDS was amplified using primers with recombination adapters. The PCR products and linearized vector were recovered using the Tiangen Universal DNA Purification and Recovery Kit. The linearized vector was ligated to the amplified gene fragment using the Tiangen EasyGeno Recombinant Cloning Kit. Genetic transformation was then performed on the highly stripe rust-susceptible wheat variety Yannong 19 (transformation method referenced Chinese Patent ZL201710422896.6). The transgenic positive plants showed increased stripe rust resistance compared to the control, indicating that the SECCE6Rv1G0452990 gene from rye can confer stripe rust resistance in wheat.

[0054] Table 4 linearization system of pWMB110 carrier

[0055] drug Volume (μl) factory Specification CutSmart Buffer 5 NEW ENGLAND 1.25ml kpnI 1 NEW ENGLAND 0.4ml SacI 1 NEW ENGLAND 0.1ml pWMB110 plasmid 10 250ng / ul <![CDATA[ddH2O]]> 33 Total 50

[0056] Incubate at 37°C for 3 hours.

[0057] The CDS gene was amplified using recombinant primers as described above. The primers used are as follows:

[0058] 2990-110-F:GGTCGACTCTAGAGGATCCCCGGGTACCGAATGGCGATGGTGCTGGATGC(SEQ IDNO:6)

[0059] 2990-110-R:TGAACGATCGGGGAAAATTCGAGCTCCTAGCTATCATGCACTTTCT(SEQ ID NO:7)

[0060] The purification and recovery of PCR products and linearized vectors were performed according to the procedures of the Tiangen Universal DNA Purification and Recovery Kit (TIANGEN catalog number: DP214-03).

[0061] The gene fragment was ligated into the linearized pWMB110 vector using a recombination method, and the specific steps were performed according to the Tiangen EasyGeno Recombinant Cloning Kit (TIANGEN catalog number: VI201-02).

[0062] Table 5. Statistical results of stripe rust inoculation of transgenic plants.

[0063]

[0064] The resistant triticale parent Rozovskaya-7 is nearly immune to stripe rust, while the susceptible parent 4100 is highly susceptible. Both the transgenic recipient material Yannong 19 and the negative transgenic material SECCE6Rv1G0452990 are nearly highly susceptible to stripe rust, while the positive transgenic material SECCE6Rv1G0452990 is nearly immune to stripe rust, indicating that SECCE6Rv1G0452990 is a stripe rust-resistant gene.

[0065] sequence

[0066] The CDS sequence (4266bp) of the rye Lo7 reference genome gene SECCE6Rv1G0452990: SEQ ID NO:1

[0067] ATGGCGATGGTGCTGGATGCGTTTGCACACTACGTGGGCCACATGCTCGCGCAGCTAGCAGCAGATGAGGTGGGGACCATGCTGGGCGTCTCCGGCGAGATCAAAAAAATGGGCGACAAGCTTCGGGATCTCAACAACTTCTTGGCTGATGCCGATAGGAGGAACATAACTGACGTGACCATCCAAGAGTGGGTGGGGCAGCTCAAGCGTGCCATGTACGAAGCTACCGACATCCTTGACCTCTGTCAGCTCAAGGCCATGGAGCGTGGATCATCTACTACTCTAGACGCAGGGTGTTTCAACCCCTTGCTCTTCTGCATGCGCAATCCCTCCCATGCTCATGAGATCGGCACCCGCATCAAAAATCTCAACAAGAGGCTCGACTCCATCAAACAACGGAGTGCTCCTTTCAACTTCGTCAATCTCGGGTCTTATGAGGATCATAACAACAACGCCCATGGCTTTCGCCATGGTAATCCAAGTCGGGAGACGGTAGGGGACTTTGACCGATCAGCTGTTGTTGGGGACAAGATTGAAGAAGATACAAGAGCACTGG

[0068] TGGCCCAAATCATGCAGACGGGAAAGGATGTCAACAATGGCATCATT

[0069] GTGGTCGCTATCGTAGGTGTTGGTGGGATCGGCAAGACCACCCTCGC

[0070] CCAGAAGGTCTTCAATGATGAGGCAATCCAAGGTGAATTCAGCAAA

[0071] AAGATATGGTTGAGTGTCAACCAAAACTTCAGTGATGTTGATCTGCT

[0072] GAGAAGGGCCATCATCGAAGCCGGAGGAGATGCCCAACCACCTGAA

[0073] AGTGCAAAGACCAGCCTTCATGAAACCCTCAAGAACATATTGATTGA

[0074] CCACAAGACCTTTCTGGTAATGGATGATGTGTGGAACCATAGAGCAT

[0075] GGGATGACGTGCTGAAAATACCCTTAGTCAATGTTGCTGGTTCAGGC

[0076] AGCCGAGTCCTCGTTACTACCAGAGAAGAAGGTGTTGCCCGAGGGG

[0077] TGAAAGCCATTTGGCCGTACCACCATATCGATACATTACTGCCTGAAG

[0078] ATGCCTGGTCATTGCTCAAGAAACAGGTATGCTCAAGGGAGTTAGAT

[0079] GAAGACCACATCAATACGCTAAAGGATATAGGACTGAAAATTATACA

[0080] GAAATGTGGTTGTTTACCAATTGCTGTTAAAGTGATGGGAGGACTCT

[0081] TGCGTGAAAGAGGGGGGCTACGTCGTGACTGGCAGCAGGTTTTGGA

[0082] TGATTCTAAATGGTCAACAACTAAAATGCCTGATGATCTCAACCACA

[0083] CAGTATACTTAAGCTATGAATATATGCCTTCTTACCTGAAGCAGTGCTT

[0084] TCTGTACTACTCTTTTCTTCCTAAAAGTAGACGTTTTCATATGGAGCA

[0085] AGTCGTGGCAATGTGGATAAGCGAAGGATTTATTCATGGAAACTCTG

[0086] GTGATTTAGAAGAATTGGGGAGAAATTACTACAAGGAGTTGGTATCT

[0087] AGGAACCTTATAGAGCCAGATAAATCATATGCTGATATATGGTTTTGC

[0088] AGCATGCATGATGTTGTTCGCTCATTTGCTCGGTATATGACTAAAGAT

[0089] GAAGCACTTGTTACTCAAGACGGAGACACTGATATGCTTGCTAAACT

[0090] TGCTTCACAAAAGTTTCTTCGGTTGTCCATAGAAACTAGCCGATCAC

[0091] AATCAGGTGAACTTGATTGGAAATCCCTACAGGCGCAGCAATCAGTG

[0092] AGAACACTGATCTCAACCATCCAGATTAAGATGATGCCTGGTGATTC

[0093] ATTGGTTACCTTTTCTAGTTTGCGGACTCTGCATATAGAATCTGCTGAT

[0094] ATGGCTGTATTGCTTGAATTGTTGCATCAACTCAAGCATGTGAGGTAT

[0095] CTAGCACTAGTAAATGCTGGTATATCTGTACTTCCAGGGAACATTGGC

[0096] AAGATGAAACTATTGCAATTCCTTGACCTTGGTGGATGTACAAAATT

[0097] GGTTAACCTTCCAGACAACATTGTGAATCTTGGCCAGCTGAGGTTAT

[0098] TTGCACTTCCCAGAGCAAGTACGGTACCTAGAGGGTTTAGTGGCCTG

[0099] ACAAATATGAGGATATTACGTATGTTTCGAGCCCACATGGATAATGAT

[0100] TGGTGCAGTTTGGACGAGTTGGGGCCTCTTTCACAGCTCAGATTTCT

[0101] TGGATTAAATGAATTAGAGAATGTATCTGCTGCCTCGTTTGCTTCTAA

[0102] TGCTAGGCTCGGCGAGAAGATGCATCTTATCACTCTACTCCTGGGTT

[0103] GCACTAGTAAGCTGGGAGATGATGGGTTCGTCAAAGAGAAGGAAGG

[0104] TGTCTCTGAGGAAGAGCAGCAACGAATTGAGAAGGTTCTTGATAAG

[0105] CTCTACCCTCCACCTGGTGTAGAAGATCTTCAAATTAGTGGGTATTTT

[0106] GGCCGGCAACTCCCGAGCTGGATAATGTCCACATCAACGGTGCCCCT

[0107] CAACAACTTGAAGACTATATTTTTTGTTGATCTGGCTTGTTGCACACA

[0108] ACTTCCCAATGGGCTGTGCCACCTCCCCAATCTGCAGTTTCTACAGG

[0109] TCTCTCGTGCTCCATGCATCAAACATGTTGGGGCTGGATTCTTGCAG

[0110] GCGGCAGCAGCTTCATTTCCAAGGTTAAACAAATTGATCTTGTTAGG

[0111] CATGGTGGAATGGGAGGAGTGGGAGTGGGAGGAGCAAGTACAAGC

[0112] CATGCCCCGTTTGGAGGAGCTTGTGCTCAATAAATGCAGACTAAGGC

[0113] ATGTTCCTCCAGGCCTTGCCTCCAATGCAAGCTCTTTGAAAATATTTG

[0114] TTCTAGTGCATGTCAAGCAACTTAGCTACATTGAGAGCTTTCCTTCTG

[0115] TTGTTGAGCTTATAGTGACTGGATGCCCAGACCTGGAGAGGATCAGC

[0116] AATCTCCCTAATCTGCAGAAGCTTACCATCCAAAACTGCCCAAAGTT

[0117] GAAGGTGCTGGAGCGTATCACTTCACTCGAGAGGCTGATCCTGAAG

[0118] GATTATACCATGGAAAAACTCCCAGAATACATGCGAGACTTAAAGCC

[0119] AAGGCATTTGCAGTTATTCTGCAGGCTATGGTTGCTCTATGCGGTAGC

[0120] CGCGGGACAATCTGGCACTGAGTGGGACAAGTTCAACCAAGTGGAG

[0121] CATGTCAAGGCATATGCACGTGATGGAGACAACCAAAGAAAATGGT

[0122] ACGTTTTGTACACGGGAGGAGACAACTACAAGCTGGATTCAAATATT

[0123] AGCAGCTCTACCATAGTTGAAGAACCCTTATCATCTTGTATGGTGGAC

[0124] CGACAAGGATTTGAGTCTCTGTACAGAATGAGAAGAAGCACCTTCA

[0125] GTTATGTTTGCAGCTTGGTGAGGATCCCATTTTTTGAAGATATGATGG

[0126] GAAGGGAGCCCACCTTTATCGATGGGAGAGTGTTGTCTTTACAAGAC

[0127] AGAGTGGCTGTCGCTCTGAGAATGCTGAATTCTGGTGACTCTCCGGT

[0128] TAACGTAGGATCCTCCCTTGGTGTGAGCGAGTCAACTTCCTTGCTGG

[0129] TAACTAAGGTGTTTGTCAAGGCCATGCTGGAGCCATTATCGCACCAC

[0130] TTCGATTGGCCAGGCTCTGCTAAAATGCAGAAGATCAAGTGCAAGTT

[0131] TGACAAGATACATGGCCTACCAAACTGTTACGGTGCTGTACATACAA

[0132] CCCAAATCACATTTGGATCACAAGTTCATGACCGTGAGGAGAATGAT

[0133] TCTGTGCTAATGCGAGTAATTGTTGATCCTGATATGAGGTTCACACAG

[0134] GTTTGGTTGGCCTCTGATCTCTTGGAGTTGGACTCTGATCTCTTGAAG

[0135] TACTATGAGGAGGATGCTGTGGTGAATGGCAGTAAGATGAAGTTATC

[0136] AGATGGCTCAGAAGATGAACAATACATAATTGGTGATGCAGGATACC

[0137] CTCTTCGTCCCTGGATCCTCACACCTTACCTGTTGGAGGATGGCCTCT

[0138] CACTCTCAGACGCCAAAGTTGAGTTCAACAGGAGACATTCTGCAGT

[0139] GACAGCTTTTGCGATAAGGGCGCTAGCAAAGTTAAAAGACACATGG

[0140] AAGTGCCTCCAGGGAGAAGGGTGGCATCCAGATAATAATGACATACT

[0141] AGATTTGACAATCTGGGTATGCTGCATGTTGCATAACATAGTGATAGA

[0142] CATGGAGAAGGAGAAGGACGAGGATCAGGAGGAAGGTGAGTATGA

[0143] GGATGAGGGCCAGGGGGAGTTGCAGCAGGTAGCAGCTGTCGGGGT

[0144] GAGGGGTGCACTGTCCCAGCACTTGATAAAGTCTGTAGAAGAGGAA

[0145] CAAGGAGCAGAAGATAAAAACAAGGAAGAAGAAGCACAACAACGG

[0146] AAGGCAACATCTCGAGGGAAGGAGAAAGTGCATGATAGCTAG

[0147] SEQ ID NO:2 CDS sequence (4248bp) of SECCE6Rv1G0452990 in hexaploid triticale Rozovskaya-7:

[0148] ATGGCGATGGTGCTGGATGCGTTTGCACACTACGTGGGCCACATGCTCGCTCAGCTAGCAGCAGATGGTGTGGGGACGATGCTGGGCGTCTCCGGCGAGATCGACAAGATGGCCGACAAGCTCCGGGACCTCAAAAACTTCTTGGCTGATGCTGATAGGAGGAACATCACCGATGAGACCGTTCAAGAGTGGGTGGGGCAGCTCAAGCGTGCCATGTATGAAGCTACCGACATCCTCGACCTCTGTCAGCTCAAGGCCATGGAGCGTGGATCATCTACTACTCTAGATGCAGGGTGTTTCAACCCCTTGCTCTTCTGCATGCGGAATCCTTCCCATGCTCATGAGATCGGCACCCGCATCAAAAAGCTCAA

[0149] CATGAGGCTCGACTCCATCAAAGAACGGAGTGCTGCTTTCAACTTCA

[0150] TCAATCTCGGGTCTTATGAGGATCATAACAACAACGCCCATGGCTTTC

[0151] GCCATGGTAATCCAAGTCGGGAGACGGTAGGGGACTTTGACCGATC

[0152] AGCTGTTGTTGGGGACAAGATTGAAGAAGACACAAGAGCACTGGTG

[0153] GCCCAAATCATGCAGACGGAAAAGGATGTCAACAATGGCATCATTGT

[0154] GGTCGCTATCGTAGGTGTTGGTGGGATCGGCAAGACCACCCTCGCCC

[0155] AGAAGGTCTTCAATGATGAGGCAATCCAAGGTGAATTCAGCAAAAA

[0156] GATATGGTTGAGCGTCAACCAAAACTTCAGTGATGTTGATCTACTGA

[0157] GAAGGGCCATCATCGAAGCCGGAGGAGATGCCCAACCACCTGAAAG

[0158] TGCAAAGACCAGCCTTCATGAAACCCTCAAGAACACATTGATTGACC

[0159] ACAAGACTTTTCTGGTAATGGATGATGTGTGGAACCATAGAGCATGG

[0160] GATGACGTGCTGAAAATACCCTTAGTCAATGTTGCTGGTTCAGGCAG

[0161] CCGAGTCCTTGTTACTACCAGAGAAGAAGGTGTTGCCCGAGGGGTG

[0162] AAAGCTATCTGGCCGTACCACCATATCGATACATTACTGCCTGAAGAT

[0163] GCCTGGTCATTGCTCAAGAAACAGGTATGCTCAAGGGAGTTAGATGA

[0164] AGAGCACATCAATACGCTAAAGGATATAGGACTGAAAATTATACAGA

[0165] AATGTGGTTGTTTACCAATTGCTGTTAAAGTGATGGGAGGACTCTTG

[0166] CATGAAAGAGGGGGGCTACGTCGTGACTGGCAGCAGGTTTTGGATG

[0167] ATTCTAAATGGTCAACAACTAAAATGCCTGATGATCTCAACCACACA

[0168] GTATACTTAAGCTATGAATATATGCCTTCTTACCTGAAGCAGTGCTTTC

[0169] TGTACTACTCTTTTCTTCCTAAAAGTAGACGTTTTCATATGGAGCAAG

[0170] TCGTGGCAATGTGGATAAGCGAAGGATTTATTCATGGAAACTCTGGT

[0171] GATTTAGAAGAATTGGGGAGAAATTACTACAAGGAGTTGGTATCTAG

[0172] GAACCTTATAGAGCCAGATAAATCATATGTTGATATATGGTTTTGCAG

[0173] CATGCATGATGTTGTTCGCTCATTTGCTCGGTATATGACTAAAGATGA

[0174] AGCACTTGTTACTCAAGATGGAGACACTGATATGCTTGCTAAACTTG

[0175] CTTCACAAAAGTTTCTTCGGTTGTCCATAGAAACTAGCCGATCACAA

[0176] TCAGGTGAACTTGATTGGAAATCCCTACAGGCGCAGCAATCAGTGA

[0177] GAACACTGATCTCAACCATCCAGATTAAGATGATGCCTGGTGATTCAT

[0178] TGGTTACCTTTTCTAGTTTGCGGACTCTGCATATAGAATCTGCTGATAT

[0179] GGCTGTATTGCTTGAATTGTTGCATCAACTCAAGCATGTGAGGTATCT

[0180] AGCACTAGTAAATGCTGGTATATCTGTACTTCCAGGGAACATTGGCA

[0181] AGATGAAACTATTGCAATTCCTTGACCTTGGTGGATGTACAAAATTG

[0182] GTTAACCTTCCAGACAACATTGTGAATCTTGGCCAGCTGAGGTTATTT

[0183] GCACTTCCCAGAGCAAGTACGGTACCTAGAGGGTTTAGTGGCCTGAC

[0184] AAATATGAGGATACTACGTATGTTTCGAGCCCACATGGATAATGATTG

[0185] GTGCAGTTTGGACGAGTTGGGGCCTCTTTCACAGCTCAGATTTCTTG

[0186] GATTAAATGAATTAGAGAATGTATCTGCTGCCTCGTTTGCTTCTAATG

[0187] CTAGGCTCGGCGAGAAGATGCATCTTATCACTCTACTCCTGGGTTGC

[0188] ACTAGTAAGCTGGGAGATGATGGGTTCGTCAAAGAGAAGGAAGGTG

[0189] TCTCTGAGGAAGAGCAGCAACGAATTGAGAAGGTTCTTGATAAGCT

[0190] CTACCCTCCCACCTGGTGTAGAAGATCTTCAAATTAGTGGGTATTTTGG

[0191] CCGGCAACTCCCGAGCTGGATAATGTCCACATCAACGGTGCCCCTCCA

[0192] ACAACTTGAAGACTATATTTTTCTTGATCTGGCTTGTTGCACACAAC

[0193] TTCCCAATGGGCTGTGCCACCTCCCCAATCTGCAGTTTCTACAGGTC

[0194] TCTCGTGCTCCATGCATCAAACATGTTGGGGCTGGATTCTTGCAGGC

[0195] GGCAGCAGCTTCATTTCCAAGGTTAAACAAATTGATCTTGTACGGCA

[0196] TGGTGGAATGGGAGGAGTGGGAGTGGGGAGGAGCAAGTACAAGCCA

[0197] TGCCCCGTTTGGAGGAGCTTGTGCTCCAATAAATGCAGACTAAGGCAT

[0198] GTTCCTCCAGGCCTTGCCTCCAATGCAAGCTCTTTGAAAATATTTGTT

[0199] CTAGTGCATGTCAAGCAACTTAGCTACATTGAGAGCTTTCCTTCTGTT

[0200] GTTGAGCTTATAGTGACTGGATGCCCTGACCTGGAGAGGATCACCAA

[0201] TCTCCCCAATCTACAGAAGCTTGACATCCAGAACTGCCCAAAGTTGA

[0202] AGGTGCTGGAGCATATCACTTCACTCGAGCGGCTAGTCCTGGAGGAT

[0203] TACACCATGGAAAGACTCCCAGAATACATGCGAGACATAAAGGCAA

[0204] GGCATTTGCAGTTATTTTGCAGGCTATGGTTGCTCTATGCGGTAGCCG

[0205] CGGGACAATCTGGCACTGAGTGGGACAAGTTCAGCCAAGTGGAGCA

[0206] TGTCAAGGCATATGCACGTGATGGAGACAACCAAAGAAAATGGTAC

[0207] GTTTCGTACACGGGAGGAGATAACTACAAGCTGGATTCAAATATTAG

[0208] CAGCTCTGCCATATTTGAAGAAACCTTATCATCTTGTATGGTGGATGC

[0209] ACAAGGATTTGACTCTCTGTACAAAATGAGAAGAAGTACCTTCAGTT

[0210] ATGTCTGCAGCTTGGTGAGGATCCCATTTTTCGAAGATATGATGGCAA

[0211] GGGAACATACCTTTGTTGATGGGAGATTGTTGTCTTTGCAAGACGGA

[0212] GTAGCTGTCGCTCTGAGGGTACTGAACTCTGGTGACTCTCCGGTTAC

[0213] CGTAGGATCCTCTCTTGGTGTGAACGAGTCAACTGTCTCCCTGGTAA

[0214] CTCAGGTGTTTGTTGAGGCCATGAAAAATCCAATAGTGCACCACTTC

[0215] AAATGGCCAACCTCTGCTAAAATGGGGAAGATCAAGCGCAAGTTTG

[0216] ACAAGATACACGGCCTGCCCAACTGCTGCGGTGTTGTACATACAACC

[0217] CAAATCACGTTTGGATCACAAGAGCGTGGCAGTGAGGAGAATGAGT

[0218] CTGTGCTAATGCGAGCCGTCGTTGATTCAGATATGAGGTTCACACAG

[0219] GTTTGGTTGGCCTCTGATCTCTTGGAGTTGGACTCTGATCTCTTGAAT

[0220] TCCTTTGAGGAGGGTGCTGTGGTGAATGGCAGTAAGCTGAAGTTATC

[0221] AAATGGCTCGGAAGTCGGAGATTACATAATTGGTGATGCAGGATACC

[0222] CTATTCGTCCCTGGATCCTCACACCTTACCTGTTAGAGGATGGCCTCC

[0223] CACTCTCAGAGGCCAAAGTTGAGTTCAACAGGAGATATTCTGCAGT

[0224] GACAGCTGTTGCGCAAAGGGCGCTAGCAAAGTTAAAAGACACATGG

[0225] AAGTGCCTCCAGGGAGAAGGGTGGCATCCAGATAATGACATGTTGA

[0226] GATGGACAATCTGGGTATGCTGCATGTTGCATAACATAGTGATAGACA

[0227] TGGAGGAGAAGGAGAAAGATGAGGAAGGTGAGGATGAGGGTCAGG

[0228] AGGAATTGCGGCAGGTAGCAGACGGGGTGAGGGGTGAACTCTCCCA

[0229] ACACTTGATAAAGTCTGTAGAGGAGGAACAAGGAGCAGAAGATAAA

[0230] AACAAGGAAGAAGAAGCACAGCAACGGAAGGCAACATCTCGAGGA

[0231] AAGGAGAAAGTGCATGATAGCTAG

[0232] SEQ ID NO:3 Amplification sequence of 6R2990-1:

[0233] CCAGAGAGCGTGTTATGGCTGCATTCATCGATTGATGCAGAGGCCGGGGGTATTCCTCCTTTTCAAAAAAAAAAAAAATAAGTACCGCGTCACCTGTATATATCTAGCTCATGCTCGTCCACGAACCGGAGATTGGGTTCATTGAGCACTAAAAGGTTGAAGGTTCACATCAATTGCCATCTTATACGGAAGAGCATATGTAGACGTGGTCCGAAATAAACTGAGAATAGTCAA

[0234] GGGTTAACCGTGTCCTCTCCCATCTTATCCTCATCTATCTTGTGCCATT

[0235] AGTATGCGTCTTGTGGCTCTCTCTCGTCTCTCCAGCCATTATTATCCTG

[0236] CTAGCTTCAGGGACAACCAGGAGTTGCTTTGAGGAGCTTAACGAGC

[0237] AGAACATCCATCCATCCCAAAGGTTAGTTACACCTAGCTAATTAAGCT

[0238] TCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCCTCTCGCATG

[0239] CTTAACTTTCACCTTACCTTTATTATTACAGTATAAGTAGCCAAGTACA

[0240] ACTAATGGCGATGGTGCTGGATGCGTTTGCACACTACGTGGGCCACA

[0241] TGCTCGCTCAGCTAGCAGCAGATGGTGTGGGGACGATGCTGGGCGT

[0242] CTCCGGCGAGATCGACAAGATGGCCGACAAGCTCCGGGACCTCAAA

[0243] AACTTCTTGGCTGATGCTGATAGGAGGAACATCACCGATGAGACCGT

[0244] TCAAGAGTGGGTGGGGCAGCTCAAGCGTGCCATGTATGAAGCTACC

[0245] GACATCCTCGACCTCTGTCAGCTCAAGGCCATGGAGCGTGGATCATC

[0246] TACTACTCTAGATGCAGGGTGTTTCAACCCCTTGCTCTTCTGCATGCG

[0247] GAATCCTTCCCATGCTCATGAGATCGGCACCCGCATCAAAAAGCTCA

[0248] ACATGAGGCTCGACTCCATCAAAGAACGGAGTGCTGCTTTCAACTTC

[0249] ATCAATCTCGGGTCTTATGAGGATCATAACAACAACGCCCATGGCTTT

[0250] CGCCATGGTAATCCAAGTCGGGAGACGGTAGGGGACTTTGACCGATC

[0251] AGCTGTTGTTGGGGACAAGATTGAAGAAGACACAAGAGCACTGGTG

[0252] GCCCAAATCATGCAGACGGAAAAGGATGTCAACAATGGCATCATTGT

[0253] GGTCGCTATCGTAGGTGTTGGTGGGATCGGCAAGACCACCCTCGCCC

[0254] AGAAGGTCTTCAATGATGAGGCAATCCAAGGTGAATTCAGCAAAAA

[0255] GATATGGTTGAGCGTCAACCAAAACTTCAGTGATGTTGATCTACTGA

[0256] GAAGGGCCATCGAAGCCGGAGGAGATGCCCAACCACCTGAAAG

[0257] TGCAAAGACCAGCCTTCATGAAACCCTCAAGAACACATTGATTGACC

[0258] ACAAGACTTTTCTGGTAATGGATGATGTGTGGAACCATAGAGCATGG

[0259] GATGACGTGCTGAAAATACCCTTAGTCAATGTTGCTGGTTCAGGCAG

[0260] CCGAGTCCTGTTACTACCAGAGAAGAAGGTGTTGCCCGAGGGGTG

[0261] AAAGCTATCTGGCCGTACCACCATATCGATACATTACTGCCTGAAGAT

[0262] GCCTGGTCATTGCTCAAGAAACAGGTTTGTACATACTATGTTTGCGTC

[0263] AATTAATATGAGTAGAAGGGAGTACTAATCTCTCAATGAAGTGAACA

[0264] ATCTATCTCCATCT

[0265] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A race-specific resistance gene, characterized in that, The nucleotide sequence of the anti-stripe rust gene is shown as SEQ ID NO:

2.

2. Expression vector, characterized in that, The expression vector comprises the anti-stripe rust gene according to claim 1.

3. A host cell characterized in that, The host cell comprises the anti-stripe rust gene according to claim 1 or the expression vector according to claim 2.

4. A method for detecting the presence or absence of a resistance to stripe rust gene in a plant to be tested, characterized in that, The method comprises detecting the presence of the anti-stripe rust gene according to claim 1 or a specific molecular marker thereof, the sequence of the specific molecular marker being shown as SEQ ID NO:

3.

5. A method for improving the ability of wheat or rye to resist stripe rust or a method for breeding wheat or rye that resists stripe rust, the method comprising introducing the anti-stripe rust gene according to claim 1 or the expression vector of claim 2 into the wheat or rye or cells of the wheat or rye.

6. A method for obtaining a plant cell or a plant carrying an anti-stripe rust gene, characterized in that, The method comprises introducing the anti-stripe rust gene according to claim 1 or the expression vector of claim 2 into the plant cell or the plant.

7. Use of the anti-stripe rust gene according to claim 1 or the expression vector according to claim 2 or the host cell according to claim 3 in improving the ability of wheat or rye to resist stripe rust or breeding wheat or rye that resists stripe rust.

8. A molecular marker for detecting the presence or absence of a plant resistance gene to stripe rust in a plant to be tested, characterized in that, The nucleotide sequence of the molecular marker is shown as SEQ ID NO:

3.

9. The method of claim 4 or 6 or the molecular marker of claim 8, wherein, The plant is wheat or rye.

10. A race to stripe disease resistance gene according to claim 1 or a method according to any one of claims 4, 5 or 6 or use according to claim 7 or molecular marker according to claim 8, characterised in that, The stripe rust is caused by physiological races CYR32, CYR33 and / or CYR 34. The nucleotide sequence of the anti-stripe rust gene is shown as SEQ ID NO:

2. The expression vector comprises the anti-stripe rust gene according to claim 1. The host cell comprises the anti-stripe rust gene according to claim 1 or the expression vector according to claim 2. The method comprises detecting the presence of the anti-stripe rust gene according to claim 1 or a specific molecular marker thereof, the sequence of the specific molecular marker being shown as SEQ ID NO:

3.

5. A method for improving the ability of wheat or rye to resist stripe rust or a method for breeding wheat or rye that resists stripe rust, the method comprising introducing the anti-stripe rust gene according to claim 1 or the expression vector of claim 2 into the wheat or rye or cells of the wheat or rye. The method comprises introducing the anti-stripe rust gene according to claim 1 or the expression vector of claim 2 into the plant cell or the plant.

7. Use of the anti-stripe rust gene according to claim 1 or the expression vector according to claim 2 or the host cell according to claim 3 in improving the ability of wheat or rye to resist stripe rust or breeding wheat or rye that resists stripe rust. The nucleotide sequence of the molecular marker is shown as SEQ ID NO:

3. The plant is wheat or rye. The stripe rust is caused by physiological races CYR32, CYR33 and / or CYR 34.

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