Two molecular markers of wheat leaf rust resistance gene Lr29 and their application

Abstract

The present invention discloses molecular markers XsdauLH3388 and Xsdau19A20A for the wheat leaf rust resistance gene Lr29 and their applications. The nucleotide sequence of the molecular marker XsdauLH3388 of the present invention is shown in SEQ ID NO.1, which is a co-dominant InDel marker; the nucleotide sequence of the molecular marker Xsdau19A20A is shown in SEQ ID NO.3, which is a co-dominant CAPS marker. The molecular markers XsdauLH3388 or Xsdau19A20A can be used to detect the wheat leaf rust resistance gene Lr29 and the genotype of plants containing the Lr29 gene. Therefore, the molecular markers of the present invention can be used for molecular marker-assisted selection breeding of leaf rust resistance wheat, accelerating the wheat breeding process and being suitable for large-scale promotion and application.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular genetics, and in particular to two molecular markers XsdauLH3388 and Xsdau19A20A of a wheat leaf rust resistance gene Lr29 and applications thereof. Background Art

[0002] Wheat is one of the most widely cultivated and important grain crops in the world. It is the staple food for approximately 35%-40% of the global population and provides approximately 20% of human energy. In recent years, my country's wheat planting area has reached approximately 360 million mu (approximately 166 million hectares), with annual production remaining at around 130 million tons.

[0003] Wheat leaf rust (Puccinia recondita f.sp. tritici) is a major disease affecting wheat production in my country. Characterized by its destructive nature, cyclical infection, and obligate parasitism, it is widespread. my country is one of the world's most endemic regions for wheat leaf rust, primarily distributed in the Yangtze River Basin, the Huanghuaihai region, and the Southwest wheat-growing areas. Major outbreaks have also occurred in North China and Northeast China, causing severe losses. Wheat leaf rust primarily damages wheat leaves, but can also infect stems, leaf sheaths, and ears. During its onset, rust absorbs nutrients from the leaves, destroying cell walls and reducing photosynthesis, leading to a decrease in thousand-kernel weight and typically reducing yield by 5% to 15%. With global warming, climatic conditions are becoming increasingly favorable for the occurrence and spread of wheat leaf rust. If timely countermeasures are not taken, the damage could be even greater in the future. Therefore, accelerating the selection and development of resistant varieties with durable, stable, and broad-spectrum resistance is a crucial measure for preventing and controlling the spread of the disease and is crucial for ensuring the safety of wheat production in my country.

[0004] The wheat family comprises approximately 350 to 500 species, widely distributed throughout the world. Wheat relatives contain many excellent resistance genes and serve as a gene pool for the genetic improvement of common wheat. Introducing beneficial genes from wheat relatives into common wheat through breeding and modern biotechnology is currently one of the main methods for improving various wheat traits. The leaf rust resistance gene Lr29 originates from a wild relative of wheat, Thinopyrum longissima, and is a dominant gene for leaf rust resistance throughout the entire growth period. In the context of common wheat, Lr29 is located on the 7E / 7D translocation chromosome, with the translocation length exceeding the short arm of chromosome 7D. Lr29 is highly resistant to the leaf rust race prevalent in my country and is not linked to other adverse agronomic traits, making it of great value for utilization.

[0005] Traditional breeding methods rely on phenotypic selection, which is time-consuming, labor-intensive, and unreliable. In contrast, genotyping using molecular markers offers significant advantages. Genotypes are determined by the variety's inherent genetic characteristics and are unaffected by environmental influences and time constraints. Therefore, this method is highly accurate and can be used to identify plants early in their development.

[0006] Co-dominant molecular markers can not only detect whether a sample carries the target gene, but also determine whether the target gene is homozygous or heterozygous. Using the co-dominant molecular marker of the Lr29 gene to identify the genotype will greatly facilitate wheat breeding. It can not only predict whether wheat is leaf rust-resistant, but also facilitate molecular marker-assisted selection breeding of leaf rust-resistant wheat, potentially yielding wheat homozygous for the leaf rust resistance gene.

[0007] There are currently no reports on co-dominant molecular markers for the wheat leaf rust resistance gene Lr29, and there is an urgent need to develop such markers to accelerate the breeding process of leaf rust-resistant wheat. Summary of the Invention

[0008] In response to the above-mentioned prior art, the present invention aims to provide two co-dominant molecular markers for the wheat leaf rust resistance gene Lr29. These markers can be used to detect the wheat leaf rust resistance gene Lr29 and the genotype of plants containing the Lr29 gene. Therefore, the molecular markers of the present invention can be used for marker-assisted selection breeding of leaf rust-resistant wheat, accelerating the wheat breeding process and making them suitable for large-scale promotion and application.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] In a first aspect of the present invention, two co-dominant molecular markers of the wheat leaf rust resistance gene Lr29 are provided, wherein the two co-linear molecular markers are an InDel marker and a CAPS marker.

[0011] The InDel marker is a deletion / insertion polymorphism, located at 379323570-379324596 bp of chromosome 7 (7D) of the Chinese Spring reference genome v1.0, and is named XsdauLH3388; its nucleotide sequence is shown in SEQ ID NO.1.

[0012] The CAPS marker is an enzyme-amplified polymorphism located at 182832530-182833169 bp of chromosome 7 (7D) of the Chinese Spring reference genome v1.0, and is named Xsdau19A20A; its nucleotide sequence is shown in SEQ ID NO.3.

[0013] In a second aspect of the present invention, two sets of primers are provided for amplifying the aforementioned molecular markers. The primer sequences for the molecular marker XsdauLH3388 are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively; and the primer sequences for the molecular marker Xsdau19A20A are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively. Specifically, the following are:

[0014] XsdauLH3388-F: 5′-GGGCTGACAGATGGTTTTGCT-3′; (SEQ ID NO.5)

[0015] XsdauLH3388-R: 5′-TGGCTCTGATGGAAGGGTGT-3′; (SEQ ID NO.6)

[0016] Xsdau19A: 5′-CAATTTTGTTCTATTTCTGTCCAG-3′; (SEQ ID NO.7)

[0017] Xsdau20A: 5′-ATATGCCCACTTGAATCACAGAG-3′. (SEQ ID NO.8)

[0018] In a third aspect, the present invention provides a kit for detecting the above-mentioned molecular markers, wherein the kit comprises the primers shown in SEQ ID NO.5 and SEQ ID NO.6 or the primers shown in SEQ ID NO.7 and SEQ ID NO.8.

[0019] A fourth aspect of the present invention provides the use of the above-mentioned molecular marker, primer pair or kit in at least one of the following 1)-4):

[0020] 1) Detection of the wheat leaf rust resistance gene Lr29;

[0021] 2) Early screening of wheat strains resistant to leaf rust;

[0022] 3) Identification of plant genotypes containing the Lr29 gene;

[0023] 4) Breeding of wheat resistant to leaf rust.

[0024] A fifth aspect of the present invention provides a method for detecting the wheat leaf rust resistance gene Lr29, comprising the following steps:

[0025] Extracting genomic DNA from the wheat to be tested and using it as a template, performing PCR amplification using the primers shown in SEQ ID NO. 5 and SEQ ID NO. 6 to obtain a first amplification product; performing electrophoresis on the first amplification product, and if a band of 923 bp in length is obtained, the wheat to be tested contains the leaf rust resistance gene Lr29;

[0026] Alternatively, genomic DNA of the wheat to be tested is extracted and used as a template, and PCR amplification is performed using the primers shown in SEQ ID NO. 7 and SEQ ID NO. 8 to obtain a second amplification product; the second amplification product is digested with Hpy188Ⅰ restriction endonuclease, and the amplification product after digestion is detected by electrophoresis. If a band of 140 bp in length is obtained, the wheat to be tested contains the leaf rust resistance gene Lr29.

[0027] Preferably, the reaction system for PCR amplification is: 1 μL of 100 ng / μL template DNA, 7.5 μL of 2x Master Mix, 0.5 μL each of 10 μmol / L forward and reverse primers, and 5.5 μL of ddH2O;

[0028] The reaction conditions for PCR amplification were as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 54°C for 15 s, extension at 72°C for 10 s, 38 cycles, post-extension at 72°C for 5 min, and storage at 15°C;

[0029] Preferably, the enzyme digestion reaction system is: 10 μL PCR product, 0.3 μL 10 U / ml Hpy188Ⅰ, 1.9 μL 10xCutsmart, 1.8 μL ddH2O; the enzyme digestion reaction conditions are: 37°C water bath for 3 hours.

[0030] A sixth aspect of the present invention provides a method for identifying the genotype of a plant containing the Lr29 gene, comprising the following steps:

[0031] Extract genomic DNA from the wheat to be tested and use it as a template. Perform PCR amplification using the primers shown in SEQ ID NO. 5 and SEQ ID NO. 6 to obtain a first amplified product. Perform electrophoresis on the first amplified product. If a 923 bp band is obtained in addition to an 852 bp non-specific amplified band, the wheat to be tested is of the Lr29 homozygous genotype. If a 1027 bp band is obtained, it indicates the lr29 homozygous genotype. If both 1027 bp and 923 bp bands are present, it indicates the Lr29lr29 heterozygous genotype.

[0032] Alternatively, genomic DNA of the wheat to be tested is extracted and used as a template, and PCR amplification is performed using the primers shown in SEQ ID NO.7 and SEQ ID NO.8 to obtain a second amplification product; the second amplification product is digested with Hpy188Ⅰ restriction endonuclease, and the amplification products after digestion are detected by electrophoresis. If a band of 140 bp in length is obtained in addition to a non-specific amplification band of 353 bp, the wheat to be tested is of the Lr29 homozygous genotype; if a band of 287 bp in length is obtained, it represents the lr29 homozygous genotype; if both 140 bp and 287 bp bands appear, it represents the Lr29 lr29 heterozygous genotype.

[0033] Preferably, the reaction system for PCR amplification is: 1 μL of 100 ng / μL template DNA, 7.5 μL of 2x Master Mix, 0.5 μL each of 10 μmol / L forward and reverse primers, and 5.5 μL of ddH2O;

[0034] The reaction conditions for PCR amplification were as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 54°C for 15 s, extension at 72°C for 10 s, 38 cycles, post-extension at 72°C for 5 min, and storage at 15°C.

[0035] Preferably, the enzyme digestion reaction system is: 10 μL PCR product, 0.3 μL 10 U / ml Hpy188Ⅰ, 1.9 μL 10xCutsmart, 1.8 μL ddH2O; the enzyme digestion reaction conditions are: 37°C water bath for 3 hours.

[0036] In the electrophoresis detection bands of the first amplification product, the nucleotide sequence corresponding to the band with a length of 923 bp is shown as SEQ ID NO.1; the nucleotide sequence corresponding to the band with a length of 1027 bp is shown as SEQ ID NO.2.

[0037] The nucleotide sequence of the second amplified product is shown in SEQ ID NO. 3 or SEQ ID NO. 4. If the nucleotide sequence of the second amplified product is shown in SEQ ID NO. 3, two 140 bp bands and a 353 bp band will appear after enzyme digestion, and the result of electrophoresis after enzyme digestion will be a 353 bp band and a 140 bp band; if the nucleotide sequence of the second amplified product is shown in SEQ ID NO. 4, a 287 bp band and a 353 bp band will appear after enzyme digestion, and the result of electrophoresis after enzyme digestion will be a 353 bp band and a 287 bp band.

[0038] Beneficial effects of the present invention:

[0039] 1) The present invention provides PCR primers for amplifying the wheat leaf rust resistance gene Lr29. The primers are stable and amplify bands with high specificity. These markers are either co-dominant InDel markers or co-dominant CPAS markers, and can accurately identify the three genotypes of Lr29Lr29, Lr29lr29, and lr29lr29, offering significant advantages over currently available dominant markers.

[0040] 2) The present invention provides the use of the two pairs of primers in screening wheat for leaf rust resistance. PCR is used to screen wheat containing the leaf rust resistance gene Lr29. Positive plants are wheat containing the Lr29 gene and showing a 923bp or 140bp band in the amplification results. Negative plants are wheat containing no 923bp or 140bp band in the amplification results and no Lr29 gene. Using the primers to detect the molecular markers XsdauLH3388 or Xsdau19A20A, wheat leaf rust resistance traits can be effectively selected, accelerating the breeding process of leaf rust resistance wheat. The marker detection is simple, fast, low-cost, and highly practical, making it suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : Phenotypic identification of wheat leaf rust in three varieties: Lr29NIL (left), Delibab (middle), and CB037 (right).

[0042] Figure 2 : Amplification pattern of molecular marker XsdauLH3388 and enzyme digestion pattern of Xsdau19A20A in Example 2; In the figure, A) electrophoresis pattern of PCR amplification of molecular marker XsdauLH3388; B) electrophoresis pattern of PCR product of molecular marker Xsdau19A20A after enzyme digestion.

[0043] Figure 3 In Example 3, molecular markers XsdauLH3388 (A) and Xsdau19A20A (B) were used to detect disease-resistant and disease-susceptible strains of Lr29NIL / Delibab F4.

[0044] Figure 4 : Phenotypes of disease-resistant and susceptible strains of the Lr29NIL / Delibab combination F4 in Example 3. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0046] As described in the Background Technology section, the leaf rust resistance gene Lr29 originates from a closely related species of wheat, Thinopyrum longipedum. It is a dominant, full-season leaf rust resistance gene that confers high resistance to the prevalent leaf rust races in my country and is unlinked to other undesirable agronomic traits, making it a highly effective leaf rust resistance gene. The development and research of molecular markers for the leaf rust resistance gene Lr29 is of great significance for marker-assisted breeding of leaf rust-resistant wheat.

[0047] There are few reports on molecular markers for the leaf rust resistance gene Lr29, and all of them are early random amplified polymorphic DNA (RAPD). RAPD has poor repeatability and instability, which limits the application of the leaf rust resistance gene Lr29 in wheat breeding.

[0048] Insertion-deletion (InDel) refers to the insertion or deletion of nucleotide fragments of varying sizes at the same genomic site between closely related species or different individuals of the same species. This refers to the insertion or deletion of one or more bases at a site in a sequence compared to another homologous sequence. InDels occur when homologous sequence alignments create gaps. However, in most cases, the ancestral sequence is unavailable, making it difficult to determine which sequence at the gap site represents an insertion or deletion. Therefore, these mutations are generally referred to as insertion / deletion mutations. InDel markers are a PCR amplification technique and are essentially length polymorphism markers.

[0049] Cleaved Amplified Polymorphism Sequences (CAPS) utilizes the DNA sequence of a known locus to design a specific set of PCR primers. These primers specifically amplify a DNA fragment at that locus. The resulting amplified product is then cleaved with a specific restriction endonuclease, and the cleaved fragments are separated by gel electrophoresis. CAPS labeling, also a PCR amplification technique, reveals information about the restriction endonuclease length variation of specific PCR fragments.

[0050] Because the Lr29 gene is located on the wheat-Thinopecten elongatum translocation chromosome, involving a very long chromosomal segment, it cannot properly associate and exchange with common wheat chromosomes. Consequently, research on the localization of the Lr29 gene has been slow due to the lack of effective molecular markers. Furthermore, the lack of a complete genome sequence of the Lr29 gene donor material has also limited the development of molecular markers for this gene.

[0051] The present invention obtains two molecular markers linked to the Lr29 gene by resequencing the germplasm material RL6080 and utilizing genomic colinearity. One of them is a co-dominant InDel marker located at 379323570-379324596 bp of chromosome 7 (7D) of the Chinese Spring reference genome, named XsdauLH3388, and its nucleotide sequence is shown in SEQ ID NO.1. The other is a co-dominant CAPS marker located at 182832530-182833169 bp of chromosome 7 (7D) of the Chinese Spring reference genome, named Xsdau19A20A, and its nucleotide sequence is shown in SEQ ID NO.3.

[0052] In order to realize the application of the molecular marker XsdauLH3388 of the present invention in molecular marker-assisted breeding of leaf rust-resistant wheat, the present invention also developed and designed primers for amplifying the molecular marker XsdauLH3388, which are specifically as follows:

[0053] XsdauLH3388-F: 5′-GGGCTGACAGATGGTTTTGCT-3′; (SEQ ID NO.5)

[0054] XsdauLH3388-R: 5′-TGGCTCTGATGGAAGGGTGT-3′. (SEQ ID NO.6)

[0055] Xsdau19A: 5′-CAATTTTGTTCTATTTCTGTCCAG-3′; (SEQ ID NO.7)

[0056] Xsdau20A: 5′-ATATGCCCACTTGAATCACAGAG-3′. (SEQ ID NO.8)

[0057] The two pairs of primers designed in the present invention have very good specificity. The primers of the present invention can accurately and effectively perform PCR amplification on the fragment where the molecular marker is located, and then the molecular marker can be effectively detected by electrophoresis or sequencing.

[0058] Moreover, the molecular markers of the present invention are all co-dominant markers, which can not only detect whether wheat contains the Lr29 gene, but also detect whether the genotype of plants containing the Lr29 gene is Lr29Lr29 or Lrlr29. Clarifying genotypes through co-dominant molecular markers will greatly facilitate wheat breeding.

[0059] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0060] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. Experimental methods without detailed conditions were carried out according to conventional test methods or the operating instructions recommended by the supplier.

[0061] Example 1: Phenotypic Identification of Three Varieties: Lr29NIL, Delibab, and CB037

[0062] The Lr29NIL material is the Thatcher near-isogenic line RL6080, which carries the leaf rust resistance gene Lr29. CB037 and Delibab are wheat varieties that do not carry the Lr29 gene. The phenotypes were determined by inoculating with a single leaf rust race, PCJT, using a smearing method.

[0063] Leaf rust resistance phenotypes such as Figure 1 Shown are Lr29NIL, Delibab, and CB037. Lr29NIL (left) shows no reaction on the leaf surface, indicating an immune phenotype. Delibab (center) and CB037 (right) both exhibited large and numerous leaf rust spores, accompanied by satellite spores, indicating a highly susceptible phenotype. Inoculated with the same leaf rust fungus using the same method at the same location and time, it can be concluded that materials containing the Lr29 gene are immune to leaf rust.

[0064] Example 2: Design and detection of molecular markers XsdauLH3388 and Xsdau19A20A.

[0065] Through Lr29NIL plant sample collection and DNA extraction, DNA samples were obtained for resequencing. After library construction, library inspection, machine sequencing, and resequencing, a total of 6.5×10 8 Sequencing data were obtained, and 1.87×10 8 De novo assembly was performed using SPAdes-3.13.1 software, resulting in 2,380,246 contigs with a total contig length of 2.7 Gbp.

[0066] Universal primers capable of amplifying chromosomes 7A, 7B, and 7D were designed in batches. By comparing the target sequences amplified by the primers with the Lr29 NIL resequencing data, two pairs of specific primers were screened. The primers were named XsdauLH3388-F and XsdauLH3388-R (specific nucleotide sequences are shown in SEQ ID NOs. 5 and 6), and Xsdau19A and Xsdau20A (nucleotide sequences are shown in SEQ ID NOs. 7 and 8). These primers can amplify specific bands through PCR and accurately identify the genotype of the wheat leaf rust resistance gene Lr29.

[0067] DNA was extracted from leaf tissue of Lr29NIL, Delibab, CB037, and a mixed pool of disease-resistant and susceptible individuals (R-pool) from Lr29NIL / Delibab hybrids. PCR amplification was performed using 38 cycles of pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 54°C for 15 seconds, and extension at 72°C for 10 seconds. Post-extension was performed at 72°C for 5 minutes, and the cells were stored at 15°C. Electrophoresis was performed on a 1.5% agarose gel using TAE buffer at 170 V for 40 minutes.

[0068] The amplification results of specific primers XsdauLH3388-F and XsdauLH3388-R showed that the mixed pool of Lr29NIL and disease-resistant individual strains had a specific band of 923 bp in length ( Figure 2 A). The amplified products of specific primers Xsdau19A and Xsdau20A were digested with Hpy188Ⅰ. The results showed that the mixed pool of Lr29NIL and disease-resistant individual strains had a specific band of 140 bp in length ( Figure 2 B).

[0069] Example 3: The accuracy of the XsdauLH3388 and Xsdau19A20A markers in identifying wheat leaf rust resistance was confirmed using the genotypes and phenotypes of the F4 resistant and susceptible lines derived from the hybridization of Lr29NIL and Delibab.

[0070] PCR amplification was performed on the F4 disease-resistant and disease-susceptible lines, and the results were as follows: Figure 3 As shown, the 923 bp or 140 bp specific band can be repeatedly amplified in the disease-resistant strains. Therefore, the XsdauLH3388 marker or the Xsdau19A20A marker can be used to effectively identify the genotypes of disease-resistant and susceptible strains.

[0071] Using a single leaf rust race PCJT, the phenotypic identification of the disease-resistant and susceptible strains of Lr29NIL and the Lr29NIL / Delibab combination F4 was carried out by smear inoculation. Figure 4As shown, the leaves of the parental material Lr29NIL and the F4 individuals 17636, 17710, 17719, 17732, and 17738, which carry the leaf rust resistance gene Lr29, showed no reaction, indicating an immune phenotype. F4 individuals 17621, 17635, 17642, 17657, 17670, 17720, 17748, and 17759, which do not contain the leaf rust resistance gene Lr29, showed large and numerous leaf rust spores accompanied by satellite spores, indicating a highly susceptible phenotype. The leaves of Jimai 22, a control, also showed numerous leaf rust spores. The phenotypic identification results were consistent with the molecular marker detection results, indicating that both the molecular markers XsdauLH3388 and Xsdau19A20A can be used to detect leaf rust resistance in wheat.

[0072] In summary, the co-dominant molecular markers XsdauLH3388 and Xsdau19A20A for the wheat leaf rust resistance gene Lr29 of the present invention can effectively detect the wheat Lr29 gene to predict whether wheat is leaf rust resistant. They can also effectively select for leaf rust resistance in wheat, enabling molecular marker-assisted selection for leaf rust-resistant wheat, thereby accelerating the wheat breeding process. The co-dominant molecular marker detection is simple, rapid, low-cost, and highly practical, making it suitable for large-scale promotion and application.

[0073] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application. SEQUENCE LISTING <110> Shandong Agricultural University <120> Two molecular markers of wheat leaf rust resistance gene Lr29 and their application <130> 2021 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 923 <212> DNA <213> Artificial sequence <400> 1 tggctctgat ggaagggtgt cttcagctat acttaaagat ggttctgttg ttgaagctga 60 tacagttacg atctttatt tctcacatg ttcttatgct gcagtagcaa atcctaaacc 120 atgaaaatca tcgagattcc tgcatttaag ttcctactgt tggtcttggc cttattaga 180 gcacccgtta tattagatta tttaatgaca agattgac cacttagatc cacttgatat 240 tatgtttgaa agcatttga taataccaa cttatctttg aaatcctgtt agtagaaagc 300 atctagacaa cctaatccct cagaagtgaa gtagagtcta tgataggtgt gctatccttc 360 tacattttat ggttagctaa taaactagac tgacaagcaa agtcctaaat tctggaacc 420 agtaagttct ggtgtgtgat gggtagaaaa tgccgagtttt tcagtatgga ggtttcatgg 480 taatgctgat ggtggtggct ctagtggagg ggaagagagg gggagaatga ggaagtcttt 540 taaatctttg attaggaac siacttgat tctcagaat cagactcagt attgttctcg 600 ctgtccacaa actaatctgc ttagaaaccc tgctgtcgt ggttggatat tgttggcatc 660 agcctctgtt tggtgaacgg tagtattagc ccaaatgtg tgtcgtgatc aaatgtctgc 720 tgaggcagca gaagttacat acagtttt attackatcac taggagcatc agctactatt 780 tattatattt gatattatct tcttatgtac atattcttaa actgctgaaa ccgtatggat 840 tcatatcatg agatcttctt atgttgacat tacattctaa tgcaggttat tgttggtata 900 ggagcaaaac catctgtcag ccc 923 <210> 2 <211> 1027 <212> DNA <213> Artificial sequence <400> 2 tggctctgat ggaagggtgt cttcagctat acttaaagat ggttctgttg ttgaagctga 60 tacagttacg atctttattt tctcaacacg ttcttatgct gcagtaccaa atcctaaacc 120 atgaaaatca tcgagattcc tgcatttgag ttcccactgt tggtcttggc cttattaaga 180 gcacccgtta tattagatta tttaatgaca agattatgac cacttagatc cacttgatat 240 tatgttcgaa aggcatttga taataaccaa cttatctttg aaatcctctt agtagaaagc 300 atgtagacaa cctaattcct cagaagtgaa gtagagtcta tgataggtgt ctatccttct 360 actccctccg ttctaaatta ctcgtcgcag aaatggatgt atctagaact aaaatacatc 420 tagatacatc catacctgcg acaagtaatt cggaacggag ggagtacatt ttatggttag 480 ctaataaact agactgacaa gcaaagtcct aaattctgga aaccagtaag ttctggtgtg 540 tgatgggtag aaaatgccga gttttcagta tggaggtttc atggtaatgc tgatggtggt 600 ggctctagtg gaggggaaga gagggggaga atgaggaagt cttttaaatc tttgattagg 660 aaaccaactt gatttctcaa gaatcagact cagtattgtt ctcgctgtcc acaaactaat 720 ctgcttagaa accctgctgg tcgtggttgg atattgttgg cgccagcctc tgtttggtga 780 aaggtagtat tagcccaaaa tgtgtgtcgt gatcaaatgt ctgctgaggc aacagaagtt 840 acatacagtt tattatatta tcagtaggag catcagctac tatttattat atttgatatt 900 atcttcttat gtacgtattc ttaaactact gaaaccgtat ggattcatat catgagatct 960 tcttatgttg acattacaat gtaatgcagg ttattgttgg tataggagca aaaccatctg 1020 tcagccc 1027 <210> 3 <211> 633 <212> DNA <213> Artificial Sequence <400> 3 atatgcccac ttgaatcaca gagtgtttga tgccacagga aaaacaagag aattgaaaaa 60 aaaggttgaa gtggatgcta gattttcaac aaaatatagt acacatgatt tcataggaaa 120 aattcctata ggatacaatc ctatgaatca aatgaccaat gtaagaaaaa ttcctaagga 180 ctctagccct ccaaaaatcc tatgattttt tttgaatcaa aggagtcctc ggtggtttgc 240 acatggtttt atatggctaa ggatgaatgg aaatgccagc tcaacaacta acgtacggcg 300 tcaagtcctt tgtttacttc atggccatgc cctgcacgct agttgttgtg atcagatgta 360 gagggtccca ccaggatgtt ttgtgtttta aggcatgggc ctaccttcta gaagataagg 420 gagattccct agaatctttt gtagctgaag atttatgtga aaatacaact gtaaacatgt 480 gggcatatgt gtccgaagtg aaaagaaaaa ttaaaaaata gtaaaaaagt caaatacctt 540 ttgcaacaaa catggtctac cgatatactc atgtataaag tttcatgaga aaaatacttc 600 cgtggtattc tggacagaaa tagaacaaaa ttg 633 <210> 4 <211> 640 <212> DNA <213> Artificial Sequence <400> 4 atatgcccac ttgaatcaca gagtgtttga tgccacagga aaaacaagag aattgaaaaa 60 aaaggttgaa gtggatgcta gattttcaac aaaatatagt acacatgatt tcataggaaa 120 aattcctata ggatacaatc ctatgaatca aatgaccaat gtaagaaaaa ttcctaagga 180 ctctagccct ccaaaaatcc tatgattttt tttgaatcaa aggagtcctc ggtggtttgc 240 acatggtttt atatggctaa ggatgaatgg aaatgccagc tcaacaacta acgtacggcg 300 tcaagtcctt tgcttacttc atggccatgc cctgcaagtt agttgttgtg atcagatgta 360 gagggtccca ctcccaccag gatgctttgt gttttaaggc atgggcctac cttctagaag 420 ataagggaga ttccctagaa tcttttgtag ctgaagattt atgtgaaaat acaactgtaa 480 acatgtgtgc atatgtgccc gaagtgaaaa gaaaaattaa aaaaatagta aaaaggtcaa 540 ataccttttg caacaaacat ggtctaccga tatactcatg tataaagttt cacgagaaaa 600 atacttccgt ggaattctgg acagaaatag aacaaaattg 640 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <400> 5 gggctgacag atggttttgc t 21 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <400> 6 tggctctgat ggaagggtgt 20 <210> 7 <211> twenty four <212> DNA <213> Artificial sequence <400> 7 caattttgtt ctatttctgt ccag 24 <210> 8 <211> twenty three <212> DNA <213> Artificial sequence <400> 8 atatgcccac ttgaatcaca gag 23

Claims

1. A molecular marker for the wheat leaf rust resistance gene Lr29, characterized in that: The molecular markers include: a co-dominant InDel marker XsdauLH3388 and a co-dominant CAPS marker Xsdau19A20A; The co-dominant InDel marker XsdauLH3388 is located at 379323570-379324596 bp of chromosome 7 (7D) of the Chinese Spring reference genome, and the InDe1 marker is a deletion / insertion polymorphism; the co-dominant CAPS marker Xsdau19A20A is located at 182832530-182833169 bp of chromosome 7 (7D) of the Chinese Spring reference genome; the CAPS marker is an enzyme-amplified polymorphism; The nucleotide sequence of the co-dominant InDel marker XsdauLH3388 is shown in SEQ ID NO.1; the nucleotide sequence of the co-dominant CAPS marker Xsdau19A20A is shown in SEQ ID NO.

3.

2. Use of the molecular marker according to claim 1 in at least one of the following 1) to 4): 1) Detection of the wheat leaf rust resistance gene Lr29; 2) Early screening of wheat strains resistant to leaf rust; 3) Identification of plant genotypes containing the Lr29 gene; 4) Breeding of wheat resistant to leaf rust.

3. Use of the primers for amplifying the molecular marker according to claim 1 in at least one of the following 1) to 4): 1) Detection of the wheat leaf rust resistance gene Lr29; 2) Early screening of wheat strains resistant to leaf rust; 3) Identification of plant genotypes containing the Lr29 gene; 4) Breeding of wheat for resistance to leaf rust; The primer sequences used to amplify the co-dominant InDel marker XsdauLH3388 are shown in SEQ ID NO.5 and SEQ ID NO.6; the primer sequences used to amplify the co-dominant CAPS marker Xsdau19A20A are shown in SEQ ID NO.7 and SEQ ID NO.

8.

4. A method for detecting the wheat leaf rust resistance gene Lr29, characterized in that: The following steps are involved: Extracting genomic DNA from the wheat to be tested and using it as a template, performing PCR amplification using the primers shown in SEQ ID NO. 5 and SEQ ID NO. 6 to obtain a first amplification product; performing electrophoresis on the first amplification product, and if a band of 923 bp in length is obtained, the wheat to be tested contains the leaf rust resistance gene Lr29; Alternatively, genomic DNA of the wheat to be tested is extracted and used as a template, and PCR amplification is performed using the primers shown in SEQ ID NO. 7 and SEQ ID NO. 8 to obtain a second amplification product; the second amplification product is digested with Hpy188Ⅰ restriction endonuclease, and the amplification product after digestion is detected by electrophoresis. If a band with a sequence length of 140 bp is obtained, the wheat to be tested contains the leaf rust resistance gene Lr29.

5. The detection method according to claim 4, characterized in that The reaction system for PCR amplification was as follows: 1 μL of 100 ng / μL template DNA, 7.5 μL of 2x Master Mix, 0.5 μL each of 10 μmol / L forward primer and reverse primer, and 5.5 μL of ddH2O; The reaction conditions of PCR amplification were as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 54°C for 15 s, extension at 72°C for 10 s, 38 cycles, post-extension at 72°C for 5 min, and storage at 15°C.

6. The detection method according to claim 4, characterized in that The enzyme digestion reaction system was as follows: 10 μL PCR product, 0.3 μL 10 U / ml Hpy188Ⅰ, 1.9 μL 10xCutsmart, and 1.8 μL ddH2O; The reaction conditions for enzyme digestion were: 37°C water bath for 3 h.

7. A method for identifying the genotype of a plant containing the Lr29 gene, characterized in that: The following steps are involved: Extract genomic DNA from the wheat to be tested and use it as a template. Perform PCR amplification using the primers shown in SEQ ID NO. 5 and SEQ ID NO. 6 to obtain a first amplified product. Perform electrophoresis on the first amplified product. If a 923 bp band is obtained in addition to an 852 bp non-specific amplified band, the wheat to be tested is of the Lr29 homozygous genotype. If a 1027 bp band is obtained, it indicates the lr29 homozygous genotype. If both 1027 bp and 923 bp bands are present, it indicates the Lr29 lr29 heterozygous genotype. Alternatively, genomic DNA of the wheat to be tested is extracted and used as a template, and PCR amplification is performed using the primers shown in SEQ ID NO.7 and SEQ ID NO.8 to obtain a second amplification product; the second amplification product is digested with Hpy188Ⅰ restriction endonuclease, and the amplification products after digestion are detected by electrophoresis. If a band of 140 bp in length is obtained in addition to a non-specific amplification band of 353 bp, the wheat to be tested is of the Lr29 homozygous genotype; if a band of 287 bp in length is obtained, it represents the lr29 homozygous genotype; if both 140 bp and 287 bp bands appear, it represents the Lr29 lr29 heterozygous genotype.

8. The method according to claim 7, characterized in that In the electrophoresis detection bands of the first amplification product, the nucleotide sequence corresponding to the band with a length of 923 bp is shown as SEQ ID NO.1; the nucleotide sequence corresponding to the band with a length of 1027 bp is shown as SEQ ID NO.2.

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