Wheat stripe rust resistant protein YR26 and its encoding gene and application

Abstract

The application belongs to the technical field of genetic engineering, and relates to a wheat stripe rust resistance protein YR26, a coding gene thereof and application. The wheat stripe rust resistance protein YR26 provided by the application has an amino acid sequence as shown in SEQ ID NO:1, and is encoded by a gene YR26 The coding sequence of the gene YR26 Is shown in SEQ ID NO:2. The sequence of the gene YR26 Is isolated for the first time, and the disease resistance function thereof is verified by various molecular biology means such as mutants, gene editing, transgenesis, gene silencing and the like.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a wheat stripe rust resistance protein YR26, its encoding gene, and its applications. Background Technology

[0002] Wheat stripe rust is caused by the wheat-specific strain of *Strombus styracifolius* (Strombus styracifolius). Puccinia striiformis f.sp. tritici , Pst Stripe rust, a fungal disease caused by stripe rust, seriously endangers wheat production. From different perspectives, including the timing of resistance manifestation, specialization to physiological races, intensity of resistance, sensitivity to temperature, number of genes controlling resistance, and duration of resistance, wheat resistance to stripe rust can generally be divided into two types: all-stage resistance and adult-plant resistance. All-stage resistance (ASR), also known as seedling resistance, is characterized by resistance in the seedling stage that persists throughout the entire growth period, exhibiting an immune or hypersensitive reaction (HR) to stripe rust. It is generally a qualitative trait, controlled by a single gene, and easily applied in breeding. Adult-plant resistance (APR) involves susceptibility to stripe rust in the seedling stage, but resistance develops into adult plants, gradually increasing with growth. APR resistance often exhibits quantitative genetic characteristics, controlled by multiple genes.

[0003] Lupton and Macer (1962) named wheat stripe rust resistance genes using the Yr (Yellow rust resistance) designation (Lupton and Macer 1962), and this naming method has been used ever since. To date, a total of 87 officially named stripe rust resistance genes have been discovered. Yr1 - Yr87 ) and more than 400 resistance QTL (quantitative traitlocus) sites, that is, however only Yr5 , Yr7 , YrSP , Yr9 , Yr15 , Yr27 , Yr28 (YrAS2388) YrU1 , Yr10 / YrNAM、 Yr84 , Yr87 and APR resistance gene Yr18 , Yr36 and Yr46 Only a few genes have been cloned, which is insufficient to meet the needs of actual production. Summary of the Invention

[0004] This invention separates the image using a map-based cloning method. Yr26 The gene sequence was obtained, and its disease-resistant function was verified using various molecular biology techniques such as mutants, gene editing, transgenics, and gene silencing. The gene encodes a predicted transmembrane protein, and no homologous gene or amino acid sequence was found in any known database, making it a completely new type of disease-resistant gene.

[0005] To ensure a complete and unambiguous understanding of the technical solution of this invention, it should be noted that the protein YR26 of this invention is represented by "YR26" in non-italicized font, and the gene... YR26 Use italic font YR26 This indicates that, of course, those skilled in the art can clearly and completely understand the meaning and description of the relevant genes and their encoded proteins based on the description in this invention.

[0006] On one hand, the present invention provides a method for breeding wheat varieties resistant to stripe rust, comprising: increasing the activity and / or content of protein YR26 in wheat, or overexpressing the gene in wheat. YR26 This enhances wheat's resistance to stripe rust; the gene... YR26 The protein YR26 is encoded, and the amino acid sequence of the protein YR26 is shown in SEQ ID NO: 1.

[0007] SEQ ID NO: 1 is as follows:

[0008] MNSEQTKETNSEQTTEGSVELTLENYEEFLSYKFDSDVRPCSTEINSQQTTEERLDYPNYKPGLLSSLLNEINSEQTTEERRMELDDYINYEAARLKLPEWLRNGRVPLYEMPTSYPDAD GNIISQVQPYFVTPPGGSNAVPVEPNHGTTGVGEGTDADILQTIQIPRIHAFQLIGAQVAARVRPSFFRAVYENMYSEASSGGGDGLGVEWNDIGQAYIRAQVAEHARRSTFEAVFGYINE TMYAEASSGGGDGLGVAWNDIARAYIRAQAAAHFRPSAFGAGFGHMNETMSAEASPGGGNGLGVEWNDDMLPALLRSSGRAIREHGGSQDFLVDEAGGRFLAIHIHPPQQGDRDQGLVGA ALGTQDGPGDLARQALPSVIGGLVPLCFNLLLDDSILKADSSPGYIKAAAAAGLGLVTAFTFLGVASKKRAKAAARVVASASTAAVSIAVVYQVSDNAYIKCVCGIMGCVATLATMAIDW.

[0009] Furthermore, in the method provided by this invention, the gene YR26 The encoded sequence is shown in SEQ ID NO: 2.

[0010] SEQ ID NO: 2 is as follows:

[0011] Gene YR26 The full-length cDNA sequence is as follows:

[0012] Furthermore, in the method provided by this invention, increasing the activity and / or content of protein YR26 in wheat includes introducing an overexpression vector into wheat, the overexpression vector containing the gene. YR26 .

[0013] On the other hand, the present invention provides the protein YR26 or the gene YR26 In its application to alter wheat resistance to stripe rust, the amino acid sequence of the protein YR26 is shown in SEQ ID NO: 1, and the gene... YR26 Encoding the protein YR26; by increasing the activity and / or content of the protein YR26 in wheat, or by overexpressing the gene in wheat. YR26 This improves wheat's resistance to stripe rust.

[0014] Furthermore, in the application provided by this invention, the gene YR26 The encoded sequence is shown in SEQ ID NO: 2.

[0015] On the other hand, the present invention provides a method for detecting whether wheat contains a stripe rust resistance gene. YR26 The method involves using the molecular detection marker DM2 to detect the wheat sample under test; the molecular detection marker DM2 is amplified and detected by a primer set containing the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4; if the product shown in SEQ ID NO: 5 is obtained by amplification, it indicates that the wheat sample under test contains the gene. YR26 If the product cannot be amplified, it indicates that the gene is not present. YR26 .

[0016] SEQ ID NO: 3 is as follows: AGTAGGGTGAGAAAGTTATAGGGTT.

[0017] SEQ ID NO: 4 is as follows: TAATAAGTTGAAGCATAGCGGCACA.

[0018] SEQ ID NO: 5 is as follows: AGTAGGGTGAGAAAGTTATAGGGTTTTTTCTAACAAGGCACACTCTCGTCAAAAAATAACAAGGCACACTAGCAGGCGTCCAAGAAAACCCAACATGAGCAGAAGGAGGGCGGTTAGGCCTTGTACAATGGGAGGTGCTTAGAAAAGGTGTTTAGAGAAATAAACCAAGCTTTCTTTAAGCACCGGTATTTATTTGTACAGGAGAGACGCTTAAATAGACACCGGTG CTTTAGAAAAACTCGGTTTATTTTTCAAGCACCTCTCTCCATTGTACAAGGCCTTACAGCCTTAACTAGTTAATTTCTTGCTCAATGCTCAGCCCCGACAACGCTTTAACTAGTTACAGGGATTAACTAATTTCCATTTCTTCCAGGATGGTCCTGGTGATCTTGCTCGGCAGGCTCTGCCCTCGGTAATTGGTGGTCTTGTGCCGCTATGCTTCAACTTATTA.

[0019] Furthermore, in the method provided by this invention, the molecular detection marker DM2 is used to detect whether a plant sample contains a gene. YR26 The gene YR26 The encoded sequence is shown in SEQ ID NO: 2.

[0020] On the other hand, the present invention provides the application of the functional KASP molecular marker KYR26A of the wheat stripe rust resistance gene Yr26 in wheat molecular-assisted breeding. The functional KASP molecular marker KYR26A is designed from the gene Yr26-specific SNP site T619C. The SNP site T619C is located at position 101 of the nucleotide sequence shown in SEQ ID NO: 6, and the polymorphism is T / C.

[0021] SEQ ID NO: 6 is as follows: TCATGTCTTAGAAATGATTTGCAGTTACTATCCAGCCTTCTCAACGAGATTAACTCTGAACAAACAACGGAGGAGAGGCGCATGGAGCTCGACGACTACATAAATTACGAGGCAGCAGTAAGTTGGTCCCAATTATATCAAGCGTTGTTCATTTCTTTTTGAGAGAAAAAAAAGTACTACTAGTACTAGTAGTACGTTCTA.

[0022] Furthermore, in the application provided by the present invention, the primers used to amplify the functional KASP molecular marker KYR26A include the susceptibility site forward primer KYR26A-FAM, the resistance site forward primer KYR26A-HEX, and the common reverse primer KYR26A-Common. The nucleotide sequence of the positive primer KYR26A-FAM for the infection site is shown in SEQ ID NO: 7; The nucleotide sequence of the positive primer KYR26A-HEX for the disease resistance site is shown in SEQ ID NO: 8; The nucleotide sequence of the common reverse primer KYR26A-Common is shown in SEQ ID NO: 9.

[0023] SEQ ID NO: 7 is as follows: ATGGAGCTCGACGACTACAT.

[0024] SEQ ID NO: 8 is as follows: ATGGAGCTCGACGACTACAC.

[0025] SEQ ID NO: 9 is as follows: TGGGACCAACTTACTGCTGC.

[0026] In addition, the present invention provides a method for screening wheat germplasm resources containing the stripe rust resistance gene Yr26, including genotyping the wheat material to be tested using the above-mentioned functional KASP molecular marker KYR26A, and determining whether the wheat material to be tested contains the stripe rust resistance gene Yr26 based on the genotyping results.

[0027] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: For the first time, a key gene for wheat resistance to stripe rust has been successfully cloned and fully identified. YR26 The discovery of the gene and its encoded protein overcame the previous limitation that this gene could only be indirectly selected through linkage markers. By comprehensively utilizing various molecular biology techniques, including mutant creation, gene editing knockout, transgenic overexpression, and virus-induced gene silencing, the study systematically demonstrated that… YR26 This is a functional gene controlling wheat resistance to stripe rust, providing a novel transmembrane protein clue for research on disease resistance mechanisms. Based on this, this invention further developed a specific PCR marker. DM2 And based on the functional SNP site-specific KASP marker KYR26A, it was achieved that... YR26Precise and efficient gene detection can be widely applied to germplasm resource screening, breeding progeny selection, and molecular marker-assisted breeding, significantly improving the efficiency and accuracy of disease-resistant variety breeding. This not only provides key genes and practical technical tools with independent intellectual property rights to solve the problem of insufficient wheat stripe rust resistance sources, but also lays a solid material and methodological foundation for improving wheat disease resistance through biotechnology. Attached Figure Description

[0028] To more clearly illustrate the technical solution of the present invention, the accompanying drawings involved in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the description are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 for Yr26 A schematic diagram of the gene cloning process. In the diagram, a shows the location of Yr26 on wheat chromosome 1B; b shows the high-density genetic map of Yr26; c shows the 92R137 BAC contiguous group of the Yr26 location region; d shows the physical map and gene annotation of the Yr26 location region; e shows six independent EMS-susceptible mutants of 92R137; and f shows the mutation location and type of the six EMS-susceptible mutants in the Yr26 candidate gene G4.

[0030] Figure 2 For silence Yr26 Experimental diagram showing the reduction of stripe rust resistance in wheat plant 92R137 by gene silencing. In the diagram, a is a schematic representation of the location of the DNA fragment used for silencing the Yr26 candidate gene G4; b shows the resistance of G4 gene-silenced plants to stripe rust; and c shows the expression level of the G4 gene in the silenced plants.

[0031] Figure 3 This diagram illustrates the experiment in which wheat plant 92R137, with the Yr26 gene knocked out through gene editing, lost its resistance to stripe rust. In the diagram, a shows the location and sequence information of the guide sequence used for knocking out the Yr26 candidate gene G4; b shows the stripe rust resistance of the G4 gene knockout plants; and c shows the gene sequence variation in the G4 gene knockout plants.

[0032] Figure 4 This diagram illustrates an experiment demonstrating how the transgenic Yr26 gene enhances the stripe rust resistance of Fielder wheat. In this diagram, a is a schematic representation of the G4 transgenic vector for the Yr26 candidate gene; b shows the resistance of the G4 transgenic plant to stripe rust.

[0033] Figure 5 For molecular detection labeling DM2Experimental diagrams illustrating the development and application of DM2. Figure a shows the location of the Yr26-specific molecular marker DM2 on the gene; figure b shows the genotyping results of the DM2 marker in different wheat varieties.

[0034] Figure 6 The results of molecular detection of 224 wheat stripe rust resistant germplasm resources using Yr26-specific KASP molecular markers.

[0035] Figure 7 Genotyping results of recombinant inbred line (RIL) progeny from the 92R137 / YM158 population. Detailed Implementation

[0036] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the embodiments described below. Unless otherwise specified, the experimental methods and detection methods in each embodiment are conventional methods; the reagents and materials can be purchased commercially unless otherwise specified.

[0037] In the following examples, the terms "resistant" or "susceptible" refer to stripe rust. Stripe rust is caused by *Strombus leptospirus*, a wheat-specific strain. Unless otherwise specified, *Strombus leptospirus* refers to the physiological race CYR32 (Yr26 shows resistance) which is widely prevalent in wheat-growing areas of China. Based on phenotype, plant resistance is defined as 0. The 9-level grading system (Line et al. 1992) indicates that a score of 0 represents immunity, 1-3 represent disease resistance, 4-6 represent moderate disease resistance, and 7-9 represent susceptibility to disease.

[0038] Example 1 This embodiment provides Yr26 Obtaining the coding gene.

[0039] Yr26 was initially located on wheat chromosome 1B ( Figure 1 (a) in order to clone Yr26 Gene, using the near-isogenic wheat NIL-R (+) line of Yr26 Yr26 ) and NIL-S (- Yr26 A new F2 segregating population was constructed; 8046 F2 plants were genotyped using the Yr26 wing molecular markers WRS-435 and WRS-312 obtained in previous studies, yielding 31 recombinant single plants; SNP variations within the recombinant range were screened using parental resequencing and transcriptome data, and 10 more molecular markers were developed and genotyped on the recombinant plants to determine the location of the recombination breakpoint, and these markers were combined with data from F2 plants and their offspring F1. 2:3 The family's resistance phenotype to wheat stripe rust fungus CYR32 will... Yr26Locating the genetic interval between molecular markers N726 and N2068 within 0.002 centimoles (cM). Figure 1 (b) in the middle.

[0040] To obtain the genomic sequence information of this localization region, a bacterial artificial chromosome library (BAC) of 92R137 was screened, resulting in 23 BAC clones. These BAC clones were linked primarily to primarily to completely cover the localization region of Yr26. Figure 1 (c) These BAC clones were sequenced and assembled to obtain a full-length genome fragment of approximately 1.2 Mb (Contig02). Genome annotation of Contig02 revealed that the fragment contained seven expressed transcripts (G1~G7). Figure 1 (d) Comparing the sequence differences of seven transcripts containing Yr26 in disease-resistant wheat 92R137, NIL-R and susceptible wheat Yangmai 158, NIL-S, and Chinese Spring, G3 contains a nucleic acid mutation that can cause amino acid changes, and G4 is a disease-resistant gene unique to disease-resistant wheat, with less than 60% homology with the sequence in susceptible wheat.

[0041] In order to determine Yr26 The coding gene for Yr26 was determined, and EMS mutant libraries of 92R137 and NIL-R were created. Phenotypic identification yielded 5 and 1 independent susceptible mutants, respectively. Transcriptome sequencing of these 6 mutants was performed, and the sequencing results were aligned to Contig02. The results showed that G4 was mutated in all 6 mutant plants, while the remaining 6 transcripts showed no difference between the parents and mutants. Therefore, G4 was preliminarily identified as the coding gene for Yr26. Figure 1 d in Figure 1 e and Figure 1 (f) Four full-length mRNA sequences (IF1-IF4) of G4 were obtained using PacBio third-generation long-read sequencing technology. IF1 had the highest content among all transcriptome data, accounting for approximately 90.48%. Further analysis revealed that IF1 encodes an open reading frame (ORF8) of a protein with 481 amino acids. Based on the mutation locations in six EMS-susceptible mutants, these mutations caused premature termination of ORF8 translation, frameshifts, or amino acid changes; all were missense mutations. Figure 1 f in the middle), therefore determine ORF8 for Yr26 The encoded sequence.

[0042] The final amino acid sequence of protein YR26 is shown in SEQ ID NO: 1; Gene YR26 The encoded sequence is shown in SEQ ID NO: 2; In addition, design genes YR26 The amplification primers were 5'-TTGCTCTAACTTGTGAAAGAAAAAA-3' and 5'-AAGAAGTGCTCAAGAAAATCGCTAC-3'; Gene YR26 The cDNA amplification primers were 5'-ATGAACTCAGAACAAACAAAGG-3' and 5'-TCACCAGTCGATGGCCATTG-3'.

[0043] Example 2 This embodiment provides silence. Yr26 This study verified that wheat's resistance to stripe rust can be reduced.

[0044] Select two Yr26 Gene-coding sequence-specific fragments 1as and 2as were obtained and ligated into the BSMV:γ vector. Yr26 Gene silencing vectors BSMV:Yr26-γ1as and BSMV:Yr26-γ1as were used. After linearization and in vitro transcription, the in vitro transcription products were inoculated onto the two-leaf pages of 92R137 seedlings. After viral symptoms appeared, wheat stripe rust fungus CYR32 was inoculated onto the four-leaf pages, and the results were detected by RT-qPCR. Yr26 Silencing efficiency in virus-infected wheat, BSMV:γ vector used as a blank control, lycopene dehydrogenase gene PDS The BSMV:PDS-γ vector was used as an indicator control to show successful viral infection. Fourteen days after inoculation, the remaining candidate genes were edited using the CRISPR system to determine their function.

[0045] Compared to wheat inoculated with the BSMV:γ vector, the expression level of Yr26 in wheat inoculated with both BSMV:Yr26-γ1as and BSMV:Yr26-γ1as vectors was significantly reduced at 24 h and 144 h post-inoculation. After inoculation with stripe rust fungus 14-16, the stripe rust disease incidence in the inoculated wheat seedlings was assessed. The results showed that wheat inoculated with the BSMV:γ vector was resistant to stripe rust, while wheat inoculated with both BSMV:Yr26-γ1as and BSMV:Yr26-γ1as vectors was susceptible to stripe rust, with leaves covered with stripe rust spores. This indicates that reducing the expression level of Yr26 can decrease the resistance of wheat to stripe rust. Figure 2 ).

[0046] Example 3 This embodiment provides gene editing. Yr26 Verification that genes can reduce wheat's resistance to stripe rust.

[0047] Using CRISPR-Cas9 gene editing technology Yr26The knockout process involved selecting two Yr26-specific targets, Target1 and Target2, and first cloning them into the pENTR:sgRNA4 vector. Then, they were cloned into the Cas9-PCL4 vector via gateway LR recombination. Figure 3 (a) The constructed vector was transformed into 92R137 wheat using Agrobacterium-mediated transformation for gene editing. Positive transgenic plants were screened by PCR amplification of the Cas9 gene. DNA sequences at two target sites were amplified in the positive transgenic plants, and Hi-TOM sequencing was performed to obtain six independent g4 mutant plants. The mutants were then passaged and screened to obtain homozygous genotypes, which were then inoculated with stripe rust race CYR32. The results showed that all six mutant plants exhibited susceptibility to the disease (…). Figure 3 (b) Genotyping analysis showed that these 6 mutant bacteria had insertion / deletion mutations of 1-6 bp. Figure 3 (c) These results illustrate Yr26 Knocking out wheat will cause it to lose its resistance to stripe rust.

[0048] Example 4 This embodiment provides Yr26 Verification of the resistance of genetically modified wheat to stripe rust.

[0049] Cloning from BAC B634-10C contains 2945 bp. Yr26 A 5825 bp genomic DNA fragment consisting of the gene coding region, a 2099 bp promoter region, and a 758 bp downstream region was constructed into the pCUB vector with the strong Ubi promoter removed, and then the vector was introduced into Agrobacterium strain EHA105. Figure 4 (a) Agrobacterium colonies were identified by colony PCR. Positive colonies were picked and cultured, then used to infect immature Fielder embryos of susceptible wheat. After subculturing the Agrobacterium-infected embryos, 23 T0 transgenic seedlings were obtained. DNA and RNA were extracted from leaves and identified by PCR. All 23 transgenic plants contained a 5825 bp fragment; however, Yr26 mRNA was undetectable in 3 of these plants, indicating that Yr26 was not normally expressed in these 3 plants. Figure 4 (b) Disease resistance and transgenicity were assessed in the T1 progeny of 23 T0 transgenes. All plants containing the Yr26 transgenic fragment and with normal gene expression showed disease resistance, while plants without the transgenic fragment or with the transgenic fragment but not expressed normally showed susceptibility. Figure 4 (b) These results demonstrate that the Yr26 transgenic wheat can acquire resistance to stripe rust.

[0050] Example 5 This embodiment provides DM2 Validation of the label for molecular detection of Yr26.

[0051] Homologous sequences of Yr26 from 47 wheat reference genomes were downloaded and subjected to multiple sequence alignment analysis along with the Yr26 gene. Based on the differences between these gene sequences, the molecular marker DM2, specifically amplifying Yr26, was designed. The full-length DM2 marker is 453 bp. Figure 5 (a) DM2 can only amplify bands in wheat containing Yr26. PCR amplification of wheat varieties such as 92R137, YM158, AvsYr24NIL, Avs, Chinese spring, Fielder, Chuanmai42, Cadenza, ArinaLrFor, and PI90962 showed that DM2 can accurately distinguish wheat varieties containing Yr26 (92R137, AvsYr24NIL, and Chuanmai42) from other wheat varieties not containing Yr26. Figure 5 (b) in the text indicates that... DM2 The markers can be used to select disease-resistant wheat plants.

[0052] Example 6 This embodiment provides the functional KASP molecular marker for the wheat stripe rust resistance gene Yr26 and its application.

[0053] Using the cloned Yr26 gene sequence (SEQ ID NO: 2) and 43 Yr26 allele sequences from different wheat materials (National Genome Data Center), numbered 1 to 43 respectively, the differences between the cloned Yr26 gene sequence (SEQ ID NO: 2) and the Yr26 allele sequences from the 43 wheat materials in NCBI were compared to identify the Yr26-specific SNP site T619C. KASP primers were designed on the Polymaker website based on the nucleotide sequences flanking SNP site T619C, and PCR amplification was performed. Finally, a functional KASP molecular marker designed based on the specific SNP site T619C was selected and named... KYR26A The specific SNP site T619C is located at... Yr26 At position 619 of the coding gene sequence, the molecular marker polymorphisms are T / C.

[0054] The DNA single-stranded sequences of 100 nt before and after the SNP site T619C are shown in SEQ ID NO: 6.

[0055] Functional KASP molecular markers KYR26A The primers include forward primers for the infection site. KYR26A -FAM, positive primer for disease resistance sites KYR26A -HEX and shared reverse primer KYR26A -Common; Forward primer for infection site KYR26A The nucleotide sequence of -FAM is shown in SEQ ID NO: 7; the "GAAGGTGACCAAGTTCATGCT" at the 5' end of SEQ ID NO: 7 is the FAM fluorescent tag sequence; Forward primers for disease resistance sites KYR26A The nucleotide sequence of -HEX is shown in SEQ ID NO:8; the “GAAGGTCGGAGTCAACGGATT” at the 5' end of SEQ ID NO:8 is the HEX fluorescent tag sequence; The nucleotide sequence of the common reverse primer KASP26A-Common is shown in SEQ ID NO: 9.

[0056] Identifying whether wheat materials contain stripe rust resistance genes Yr26 The method includes the following steps: Step 1: Extract genomic DNA as a template for amplification; Genomic DNA was extracted from the wheat materials to be tested and used as amplification templates.

[0057] Step 2: Using the genomic DNA of the wheat material to be tested as a template, PCR amplification of the genomic DNA of the sample to be tested was performed using primers with the functional KASP molecular marker KYR26A to obtain the amplification product; The PCR amplification reaction system was as follows: DNA 0.2 μg, 2×KASP V4.0 Mastermix 2 μL, primer mixture 0.044 μL, ddH2O 1.956 mL.

[0058] The primer mixture was prepared by mixing the susceptibility site forward primer KYR26A-FAM, the resistance site forward primer KYR26A-HEX, the common reverse primer KYR26A-Common, and ddH2O in a volume ratio of 12:12:30:46.

[0059] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 65℃ annealing for 60 s, 10 cycles, with the annealing temperature decreasing by 0.6℃ per cycle; 94℃ denaturation for 20 s, 55℃ annealing for 60 s, 32 cycles.

[0060] Step 3: Transfer the amplification product to a microplate reader to read the fluorescence data. Then, import the fluorescence data into KlusterCaller software for analysis to obtain genotype data. The genotypes are as follows: homozygous alleles with HEX fluorescence are denoted as "Y:Y"; homozygous alleles with FAM fluorescence are denoted as "X:X"; heterozygous genotypes are denoted as "X:Y" or "Y:X"; and genotypes with deletion values ​​are denoted as "?". If the genotype is "X:X", "X:Y", or "Y:X", then the tested wheat material contains the stripe rust resistance gene. Yr26 If the genotype is "Y:X" or "?", then the wheat material being tested does not contain the stripe rust resistance gene. Yr26。

[0061] Using the above methods, molecular detection was performed on 224 wheat stripe rust resistant germplasm resources from domestic and foreign colored wheat, local varieties, varieties (lines), and some known vector varieties (lines) containing the Yr26 gene. The results showed that 15 wheat germplasm resources were consistent with the grouping results of 92R137, namely 03031-1-5-H62, Sonalika, Chuanmai 104, Lantian 05-9-4-3, Longjian 9832, Tianxuan 49, Tianxuan 61, Tianxuan 63, Tianxuan 66, Tianxuan 69, Tianxuan 73, Tianxuan 74, Tianxuan 79, Wudu 24, and Wudu 25. Figure 6 Chuanmai 104 is a derivative of Chuanmai 42. Lantian 05-9-4-3, Longjian 9832, Tianxuan 49, Tianxuan 61, Tianxuan 63, Tianxuan 66, Tianxuan 69, Tianxuan 73, Tianxuan 74, and Tianxuan 79 are stripe rust-resistant wheat varieties or high-generation lines newly bred in Gansu Province in recent years using Yr26 (92R137) as the resistance source. These wheat varieties are all considered to contain the Yr26 / YrCH42 / Yr24 gene. These results indicate... KYR26A Able to distinguish between containing and not containing Yr26 wheat, KYR26A It can be used as a functional diagnostic marker for wheat stripe rust resistance genes. Yr26 Germplasm resource screening.

[0062] Hybrid breeding is the most important breeding method for wheat. Wheat 92R137 is a stripe rust-resistant wheat variety that contains... Yr26 The gene indicates that Yangmai 158 is a wheat variety that is completely susceptible to stripe rust and does not contain it. Yr26 Genotyping of recombinant inbred line (RIL) progeny from the 92R137 / YM158 population was performed using the functional KASP molecular marker KYR26A and related methods. The results showed that RIL progeny families could be clearly divided into two types, with co-segregation of disease resistance. Figure 7KYR26A exhibited single dominant phenotype in the 92R137 / YM158 population. Wheat containing Yr26, consistent with parent 92R137, amplified FAM fluorescence, while RIL families lacking Yr26 showed no detectable FAM or HEX fluorescence, consistent with Yangmai 158, and both clustered together with the control NTC (ddH2O). These results indicate that KYR26A can replace phenotypic selection for... Yr26 Making a selection can be applied to Yr26 Molecular-assisted selection breeding can accelerate the breeding process.

[0063] The above embodiments are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but rather to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A method for detecting whether wheat contains a gene for resistance to stripe rust. YR26 The method is characterized by, The molecular marker DM2 was used to detect the wheat samples to be tested; The molecular detection marker DM2 is amplified and detected by a primer set containing the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4; If the product shown in SEQ ID NO: 5 is obtained by amplification, it indicates that the wheat sample to be tested contains the gene YR26; If the product cannot be amplified, it indicates that the gene YR26 is not present. The molecular detection marker DM2 is used to detect whether the plant sample contains the gene YR26, the coding sequence of which is shown in SEQ ID NO:

2.

2. A wheat stripe rust resistance gene Yr26 The application of the functional KASP molecular marker KYR26A detection reagent in wheat molecular-assisted breeding is characterized by, The functional KASP molecular marker KYR26A was designed from the Yr26-specific SNP site T619C; the SNP site T619C is located at position 101 of the nucleotide sequence shown in SEQ ID NO: 6, and the polymorphism is T / C. Primers used to amplify the functional KASP molecular marker KYR26A include the susceptibility site forward primer KYR26A-FAM, the resistance site forward primer KYR26A-HEX, and the common reverse primer KYR26A-Common. The nucleotide sequence of the positive primer KYR26A-FAM for the infection site is shown in SEQ ID NO: 7; The nucleotide sequence of the positive primer KYR26A-HEX for the disease resistance site is shown in SEQ ID NO: 8; The nucleotide sequence of the common reverse primer KYR26A-Common is shown in SEQ ID NO:

9.

3. A method for screening wheat germplasm resources containing the stripe rust resistance gene Yr26, characterized in that, This includes using the functional KASP molecular marker KYR26A as described in claim 2 to perform genotyping on the wheat material to be tested, and determining whether the wheat material to be tested contains the stripe rust resistance gene Yr26 based on the genotyping results; Among them, the homozygous allele genotype with HEX fluorescence is "Y:Y", the homozygous allele genotype with FAM fluorescence is "X:X", the heterozygous genotype is "X:Y" or "Y:X", and the deletion value genotype is "?". If the genotype is "X:X", "X:Y", or "Y:X", then the wheat material being tested contains the stripe rust resistance gene. Yr26 If the genotype is "Y:X" or "?", then the wheat material being tested does not contain the stripe rust resistance gene. Yr26 .

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