Molecular marker for detecting stripe rust and brown leaf rust resistant one-cause multiple-effect sites of wheat and use method of molecular marker for detecting stripe rust and brown leaf rust resistant one-cause multiple-effect sites of wheat

Abstract

The invention discloses a molecular marker for detecting stripe rust and brown leaf rust resistant one-cause multiple-effect sites of wheat and a use method, and belongs to the technical field of nucleotide polymorphism and molecular markers. The technical problem to be solved by the invention is how to quickly and accurately screen or assist in screening wheat with stripe rust or / and leaf rust resistance or / and breed or assist in breeding wheat single plants with stripe rust or / and leaf rust resistance. The invention provides a composition for detecting single nucleotide polymorphism or genotype of a specific SNP site in a wheat genome, the SNP site can be the 47th nucleotide of a DNA fragment as shown in SEQ ID NO: 1 in the wheat genome, and the type of the nucleotide can be A or T. The primer composition and the molecular marker provided by the invention assist in wheat breeding for resisting stripe rust and leaf rust in wheat disease resistance breeding, greatly shorten the breeding period and play an important role in wheat breeding.

Description

Technical Field

[0001] This application belongs to the field of nucleotide polymorphism and molecular marker technology, specifically relating to a molecular marker for detecting pleiotropic sites of resistance to stripe rust and leaf rust in wheat and its application method. Background Technology

[0002] Stripe rust and leaf rust are significant diseases affecting wheat production, and their widespread outbreaks severely impact wheat crop safety. In recent years, with global warming and the emergence of heat-resistant stripe rust fungi, the prevalence of leaf rust and stripe rust may further expand. While chemical agents are effective in controlling multiple diseases simultaneously and have reduced disease incidence to some extent, they increase production costs and cause environmental pollution. Developing and promoting varieties resistant to multiple diseases is the most economical, effective, and environmentally friendly measure for controlling wheat diseases. Summary of the Invention

[0003] The technical problem this application aims to solve is: how to rapidly and accurately screen genes related to resistance to stripe rust and / or leaf rust, compare or assist in comparing wheat resistance to stripe rust and / or leaf rust, screen or assist in screening wheat plants resistant to stripe rust and / or leaf rust, and breed or assist in breeding wheat plants resistant to stripe rust and / or leaf rust. To solve the above technical problem, this application adopts the following technical solution:

[0004] This application provides a composition for detecting single nucleotide polymorphisms or genotypes at specific SNP sites in the wheat genome. The SNP site may be the 47th nucleotide of the DNA fragment shown in SEQ ID NO:1 in the wheat genome, and the nucleotide type may be A or T. The composition may include competitive primer A, competitive primer B, and a universal primer, as shown in A1) or A2) below:

[0005] A1) The competitive primer A may be a single-stranded DNA with a nucleotide sequence as shown in positions 22-43 of SEQ ID NO:2, the competitive primer B may be a single-stranded DNA with a nucleotide sequence as shown in positions 22-43 of SEQ ID NO:3, and the universal primer may be a single-stranded DNA with a nucleotide sequence as shown in SEQ ID NO:4.

[0006] A2) The competitive primer A may be a single-stranded DNA with a nucleotide sequence as shown in SEQ ID NO:2, the competitive primer B may be a single-stranded DNA with a nucleotide sequence as shown in SEQ ID NO:3, and the universal primer may be a single-stranded DNA with a nucleotide sequence as shown in SEQ ID NO:4.

[0007] In the composition described in this application, the ratio of the amounts of the competitive primer A, the competitive primer B, and the universal primer can be 2:2:5.

[0008] Reagents containing the above composition are also protected under this application.

[0009] Kits containing the above-described compositions and / or reagents are also protected under this application.

[0010] This application also provides a DNA molecule, which is a DNA molecule including the above-mentioned SNP sites.

[0011] In some embodiments of this application, the DNA molecule may be a DNA molecule with a nucleotide sequence as shown in SEQ ID NO:1. The DNA molecule may also be a DNA molecule with a nucleotide sequence as shown in positions 26 to 94 of SEQ ID NO:1.

[0012] This application also provides the application of the above-mentioned SNP sites and / or DNA molecules in identifying or assisting in the identification of wheat stripe rust and / or leaf rust resistance, or / and their application as molecular markers for wheat stripe rust and / or leaf rust resistance.

[0013] This application also provides the use of the above-described compositions, reagents, kits, and / or DNA molecules in at least one of the following:

[0014] B1) Application in identifying or assisting in the identification of resistance to wheat stripe rust and / or leaf rust;

[0015] B2) Application in the preparation of products for identifying or assisting in the identification of resistance to wheat stripe rust and / or leaf rust;

[0016] B3) Application in comparing or assisting in comparisons of wheat stripe rust and / or leaf rust resistance;

[0017] B4) Application in the preparation of products for comparative or auxiliary comparison of resistance to wheat stripe rust and / or leaf rust;

[0018] B5) Application in identifying or assisting in the identification of superior genes for resistance to wheat stripe rust and / or leaf rust;

[0019] B6) Application in the preparation of products for the identification or auxiliary identification of superior genes for resistance to wheat stripe rust and / or leaf rust;

[0020] B7) Application in screening, breeding, assisted screening or assisted breeding of wheat plants, lines, strains or varieties containing superior genes for resistance to wheat stripe rust and / or leaf rust;

[0021] B8) Application in the preparation, screening, breeding, assisted screening or assisted breeding of wheat single plants, lines, strains or varieties containing superior genes for resistance to wheat stripe rust and / or leaf rust;

[0022] B9) Applications in wheat breeding and / or assisted breeding;

[0023] Application of B10 in the preparation of products for wheat breeding and / or assisted breeding.

[0024] In this application, the evaluation indicators for wheat breeding may include resistance to stripe rust and / or leaf rust.

[0025] In this application, the purpose of wheat breeding may be to obtain wheat with altered resistance to stripe rust and / or leaf rust, for example, to obtain wheat with improved resistance to stripe rust and / or leaf rust.

[0026] This application also provides a method for breeding single wheat plants, lines, strains, or varieties with resistance to stripe rust and / or leaf rust, the method comprising the step of selecting wheat with the genotype TT at the above-mentioned SNP locus as a parent for breeding, wherein the TT genotype represents a homozygous type of nucleotide T at the SNP locus in claim 1 in the wheat genome.

[0027] This application also provides a method for identifying or assisting in the identification of wheat resistant to stripe rust and / or leaf rust, the method comprising the step of identifying or assisting in the identification of wheat resistance to stripe rust and / or leaf rust based on the polymorphism or genotype of the above-mentioned SNP loci of the wheat to be tested, wherein the wheat to be tested with the genotype TT at the SNP locus can be or can be a candidate wheat resistant to stripe rust and / or leaf rust.

[0028] This application also provides a method for comparing or assisting in the comparison of wheat with resistance to stripe rust and / or leaf rust, the method comprising the step of comparing or assisting in the comparison of wheat resistance to stripe rust and / or leaf rust based on the polymorphism or genotype of the above-mentioned SNP loci of the wheat to be tested, wherein the resistance of the wheat to be tested with the genotype TT of the SNP locus is higher or candidate higher than that of the wheat to be tested with the genotype AA or AT of the SNP locus.

[0029] This application also provides a method for screening and removing wheat plants with relatively low resistance to stripe rust and / or leaf rust, the method comprising the steps of detecting the genotype of the above-mentioned SNP loci and removing wheat plants with the genotype of AA or AT at the SNP loci.

[0030] In this application, the TT genotype indicates that the nucleotide type of the above-mentioned SNP site in the wheat genome is homozygous for T.

[0031] In this application, the AA genotype indicates that the nucleotide type of the above-mentioned SNP site in the wheat genome is homozygous for A.

[0032] In this application, the AT genotype indicates that the nucleotide types of the above-mentioned SNP sites in the wheat genome are heterozygous for A and T.

[0033] In this application, the above-mentioned compositions, reagents and / or kits are used to detect the genotype of the above-mentioned SNP sites in the wheat genome.

[0034] In this application, the genotype of the above-mentioned SNP site is obtained by detection using the above-mentioned composition and / or the above-mentioned reagents or kits.

[0035] In this application, the method for detecting the genotype of the above-mentioned SNP sites in wheat to be tested includes using the genomic DNA of the wheat to be identified as a template, performing PCR amplification using the above-mentioned primer composition, reagent or kit to obtain PCR products; and determining the genotype of the SNP sites based on the sequencing results or fluorescence signals of the PCR products.

[0036] In this application, the 5' end of the single-stranded DNA shown in SEQ ID NO:2 and SEQ ID NO:3 is a specific adapter sequence for the fluorescent probe.

[0037] In this application, the fluorescent probes of the single-stranded DNA shown in SEQ ID NO:2 and SEQ ID NO:3 have different specific adapter sequences, and bind to fluorescent probes of different luminescent types in the reaction system.

[0038] In one embodiment of this application, the nucleotides 1-21 of the single-stranded DNA shown in SEQ ID NO:2 are specific adapter sequences for the FAM fluorescent probe, and the nucleotides 1-21 of the single-stranded DNA shown in SEQ ID NO:3 are specific adapter sequences for the HEX fluorescent probe.

[0039] The AA genotype amplification product, when combined with the FAM fluorescent probe, exhibits blue fluorescence. The TT genotype amplification product, when combined with the HEX fluorescent probe, exhibits red fluorescence. The AT genotype amplification product, when combined with both the FAM and HEX fluorescent probes, exhibits green fluorescence. In other words, wheat samples with red-fluorescent amplification products show higher or higher resistance to stripe rust and / or leaf rust than wheat samples with blue or green-fluorescent amplification products.

[0040] In this application, the SNP site is located on wheat chromosome 5B.

[0041] In this application, the wheat may be a recombinant inbred line or a homozygous line.

[0042] In this application, the stripe rust and / or leaf rust resistance may be the adult plant resistance to stripe rust and / or leaf rust of wheat.

[0043] In this application, the pathogen causing stripe rust may be at least one or at least two of the physiological races CYR32 and CYR34 of stripe rust fungus.

[0044] In this application, the pathogen causing the leaf rust disease may be at least one, two, three, or four of the physiological races of leaf rust fungi THTT, THJH, PHTH, and FHRL.

[0045] The wheat mentioned above may be any one or more of the following varieties: Jingshuang 16, Bainong 64, Jingshuang 16 / Bainong 64 RIL population, and the 322 wheat varieties (lines) shown in Table 2 of the Examples section.

[0046] The beneficial technical effects achieved by this application are as follows:

[0047] This application utilizes the Jingshuang 16 / Bainong 64 RIL population combined with a 15K microarray to locate an adult resistance locus, QYr.caas-5BL / QLr.caas-5BL, on chromosome 5BL, which provides resistance to both stripe rust and leaf rust. This locus explains 3.7–12.3% of the phenotypic variation, corresponding to a physical region of 663.7–670.1 Mb in the Chinese Spring Reference Genome (IWGSC RefSeq v1.1). The resistance allele originates from Bainong 64. Based on this, this application transforms the flanking SNP marker AX-108936241 of the QTL mapping region into the KASP marker K_AX-108936241. Experiments have shown that the KASP molecular marker K_AX-108936241 described in this application can be used to detect the genotype of the resistance locus QYr.caas-5BL / QLr.caas-5BL in adult plants resistant to both stripe rust and leaf rust, and can be used to assist in the breeding of adult wheat varieties resistant to both stripe rust and leaf rust. Attached Figure Description

[0048] Figure 1 The location of QYr.caas-5BL / QLr.caas-5BL and its linked SNP markers in the linkage map (partial) of chromosome 5B and the LOD curve of the locus.

[0049] Figure 2 The results of the detection of K_AX-108936241 in the RIL populations of Jingshuang 16, Bainong 64, and Jingshuang 16 / Bainong 64 are as follows.

[0050] Figure 3 The test results are for 322 natural varieties (lines) K_AX-108936241. Detailed Implementation

[0051] I. Terms used in this application:

[0052] Examples of resources describing many of the molecular biology-related terms used in this article can be found in the following literature: Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, Genes IX, Oxford University Press: New York, 2007.

[0053] Any references cited in this article, including, for example, all patents, published patent applications and non-patent publications, are incorporated in their entirety by reference.

[0054] For ease of understanding of this disclosure, several terms and abbreviations used herein are defined as follows:

[0055] When used in a list of two or more items, the terms "or and" or "and / or" indicate that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0056] The term "comprising" is not intended to be restrictive, but rather inclusive and implies the presence of other elements besides those listed, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "substantially consisting of". In this document, the terms "including" and "comprise" are used interchangeably.

[0057] As used herein, the terms “marker,” “genetic marker,” “nucleic acid marker,” and “molecular marker” are used interchangeably to refer to an identifiable location on a chromosome whose heredity can be monitored and / or a reagent used in methods for visualizing differences in nucleic acid sequences (present at such identifiable locations on a chromosome). Thus, in some embodiments, a marker comprises a known or detectable nucleic acid sequence. Examples of markers include, but are not limited to: genetic markers, protein composition, peptide levels, oil composition, carbohydrate composition, fatty acid composition, amino acid composition, biopolymers, starch composition, starch levels, fermentable starch, fermentation yield, fermentation efficiency (e.g., as digestibility capture at 24, 48, and / or 72 hours), energy yield, minor compounds, metabolites, morphological characteristics, and agronomic characteristics. Thus, a marker may comprise a nucleotide sequence already associated with an allele or a target allele, and this nucleotide sequence indicates the presence or absence of the target allele in a cell or organism, and / or a reagent used to visualize differences in the nucleotide sequence at one or more identifiable locations.The markers can be, but are not limited to, alleles, genes, haplotypes, restriction fragment length polymorphisms (RFLP), simple repeat sequences (SSR), random amplified polymorphic DNA (RAPD), enzyme-mediated amplified polymorphic sequences (CAPS) (Rafalski and Tingey, Trends in Genetics 9:275 (1993)), amplified fragment length polymorphisms (AFLP) (Vos et al., Nucleic Acids Res. 23:4407 (1995)), single nucleotide polymorphisms (SNP) (Brookes, Gene 234:177 (1993)), sequence characteristic amplified regions (SCAR) (Paran and Michelmore, Theor. Appl. Genet. 85:985 (1993)), and sequence-tagged sites (STS) (Onozaki et al., Euphaitica). 138:255 (2004)), single-strand conformation polymorphism (SSCP) (Orita et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 86:2766 (1989)), simple repeat sequence interval (ISSR) (Blair et al., Theor. Appl. Genet. [Theoretical and Applied Genetics] 98:780 (1999)), intertransposon amplification polymorphism (IRAP), retrotransposon microsatellite amplification polymorphism (REMAP), Kalendar et al., Theor. Appl. Genet. [Theoretical and Applied Genetics] 98:704 (1999)), or RNA cleavage products (e.g., Lynx tags). Tags can be present in genomic nucleic acids or expressed nucleic acids (e.g., ESTs). The term "tag" can also refer to nucleic acids used as probes or primers (e.g., primer pairs) for amplification, hybridization, and / or detection of nucleic acid molecules according to methods well known in the art.

[0058] In this application, the PCR primers in the primer composition may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Markers include, but are not limited to, dyes; radioactive markers, such as 32p; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum via fluorescence resonance energy transfer (FRET). The marker can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moieties (positive or negative) or, optionally, charge-neutral. The marker can include nucleic acid or protein sequences or combinations thereof, provided that the sequence containing the marker is detectable. In some embodiments, nucleic acids are detected directly without labeling (e.g., direct sequence reading).

[0059] The term “gene” refers to a segment of DNA involved in the production of a polypeptide chain; it includes regions before and after the coding region (leader and tail regions) involved in the transcription / translation of the gene product and the regulation of said transcription / translation, as well as insertion sequences (introns) between individual coding regions (exons).

[0060] The term "allele" refers to one of several alternative forms of a gene or non-coding region of DNA that occupies the same location on a chromosome. The term allele can be used to describe DNA from any organism, including but not limited to bacteria, viruses, fungi, protozoa, molds, yeasts, plants, humans, non-humans, animals, and archaea.

[0061] The term "genotype" refers to the total combination of all genes in an organism. Organisms include, but are not limited to, diploids, tetraploids, or other possible polyploids. For example, in diploids, the genotype can be homozygous or heterozygous. A homozygous genotype means that the two alleles are identical, such as AA or aa. A heterozygous genotype means that the two alleles are different, such as Aa. Other polyploid genotypes can be described based on the specific allele distribution.

[0062] The term "template" refers to any nucleic acid molecule that can be used for the amplification described in this application. Non-natural double-stranded RNA or DNA can be made into double-stranded DNA and thus used as double-stranded DNA. Any double-stranded DNA or preparation containing a variety of different double-stranded DNA molecules can be used as template DNA to amplify one or more loci contained within the template DNA.

[0063] The term "primer" refers to an oligonucleotide that can be used in amplification methods such as polymerase chain reaction (PCR) to amplify a nucleotide sequence based on a polynucleotide sequence corresponding to a specific genomic sequence. At least one PCR primer used to amplify the polynucleotide sequence is sequence-specific to that sequence.

[0064] The term "amplification reaction" refers to a process used for one or more copies of nucleic acids. In embodiments, the amplification methods include, but are not limited to: polymerase chain reaction (PCR), self-sustaining sequencing reaction (SCRS), ligase chain reaction (LCRS), rapid amplification of cDNA ends, PCR and LCRS, Q-β phage amplification, strand displacement amplification, or overlap extension splicing PCR. In some embodiments, single-molecule nucleic acids are amplified, for example, by digital PCR.

[0065] II. Implementation Examples

[0066] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0067] In the quantitative experiments in the following examples, two replicate experiments were set up, and the average value of the results was taken.

[0068] The Jingshuang 16 variety in the following examples is a winter wheat variety jointly bred by the Beijing Shuangqiao Farm Science and Technology Station and the Beijing Academy of Agricultural Sciences Crop Research Institute. It is highly susceptible to powdery mildew in the field. It is described in the following literature: Wang Wanyi. Characteristics and cultivation techniques of the new winter wheat variety Jingshuang 16 [J]. Beijing Agricultural Sciences, 1984, 8:30-32.

[0069] Bainong 64, mentioned in the following examples, is a semi-winter wheat variety bred by the Wheat Breeding Center of Henan University of Science and Technology. It is characterized by disease resistance, wide adaptability, high and stable yield, and high resistance to powdery mildew in the field. It is described in the following literature: Zhao Hong, Wang Xicheng, Fan Hejun. A new wheat line with multiple resistance, wide adaptability, high quality, high yield and stable yield - Bainong 64 [J]. Henan Agricultural Sciences, 1997, 09: 41.

[0070] Mingxian 169: References: Huang Liang, Liu Taiguo, Xiao Xingzhi, et al. Evaluation of resistance to stripe rust and molecular detection of genes in 79 wheat varieties (lines) in China [J]. Chinese Agricultural Science, 2017, 50(16):3122–3134. The public can obtain it from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0071] Zhengzhou 5389: Highly susceptible to leaf rust, often used as a susceptible control. It is recorded in the following literature: Qin Jinyan, Li Zaifeng, Yan Xiaocui, et al. Molecular identification of leaf rust resistance gene in wheat disease-resistant line 5R625 [J]. Acta Agronomica Sinica, 2015, 41(04): 651-657.

[0072] The 322 wheat materials shown in Example 3 are described in the literature: Xu X, Sun D, ​​Ni Z, et al. Molecular identification and validation of four stable QTLs for adult-plant resistance to powdery mildew in Chinese wheat cultivar Bainong 64[J], Theoretical and applied genetics, 2023, 136: 232

[0073] The physiological races CYR32 and CYR34 of stripe rust are described in the following literature: Hu Chaoyue, Wang Fengtao, Lang Xiaowei, et al. Analysis of resistance genes against stripe rust in wheat to the main prevalent races of stripe rust in China [J]. Chinese Agricultural Science, 2022, 55: 491-502.

[0074] The physiological races THTT, THJH, PHTH, and FHRL of leaf rust are described in the following literature: Zhang P, Yan X, Gebrewahid TW, et al. Genome-wide association mapping of leaf rust and striperust resistance in wheat accessions using the 90K SNP array. Theoretical and applied genetics, 2021, 134: 1233-1251.

[0075] Disease severity estimation method (DS): the percentage of the total leaf area affected by disease; the smaller the DS (%) value, the higher the resistance.

[0076] Example 1: Obtaining the resistance locus QYr.caas-5BL / QLr.caas-5BL and its closely linked markers in adult plants resistant to both stripe rust and leaf rust.

[0077] 1. Test materials: Jingshuang 16, Bainong 64 and Jingshuang 16 / Bainong 64 recombinant inbred line populations (including 171 families).

[0078] 2. Field trials: The recombinant inbred line population of Jingshuang 16 / Bainong 64 (containing 171 families) was planted in Tianshui in 2020-2021 and in Tianshui, Chengdu, and Yangling in 2021-2022 for stripe rust resistance identification in the field. The parents and the susceptible control Mingxian 169 were planted every 17 rows, and the inducing seed Mingxian 169 was planted perpendicular to the planting direction of the population materials. The inducing seed was inoculated with stripe rust physiological races CYR32 and CYR34 to promote disease development. The recombinant inbred line population of Jingshuang 16 / Bainong 64 was planted in Baoding in 2020-2021 and in Baoding, Xinxiang, and Zhengzhou in 2021-2022 for leaf rust resistance identification in the field. The parents and the susceptible control Zhengzhou 5389 were planted every 17 rows, and the inducing seed Zhengzhou 5389 was planted perpendicular to the planting direction of the population materials. The inducing seed was inoculated with leaf rust physiological races THTT, THJH, PHTH, and FHRL to promote disease development. All field trials employed a completely randomized block design with two replicates, single-row plots, row lengths of 1m, row spacing of 0.25m, and 50 seeds sown per row.

[0079] 3. Field phenotypic identification: The investigation method for both stripe rust and leaf rust adopts the disease severity estimation method (DS), that is, to count the proportion (%) of spore masses on diseased leaves to the total leaf area.

[0080] 4. Genetic Map Construction: Families with marker deletion rates higher than 20% were removed. Then, Tassel V5.0 was used to remove segregating markers with no polymorphism, deletion rates higher than 20%, and maximum allele frequencies greater than 85%. The remaining polymorphic markers were optimized using the BIN function of IciMapping V4.2. Finally, JoinMap V4.0 was used to construct the genetic map, and Mapchart V2.3 was used for visualization.

[0081] 5. QTL Mapping: Complete Interval Mapping (CIM) was used in Windows QTL Cartographer 2.5 to perform QTL mapping on the Jingshuang 16 / Bainong 64RIL population. After 1000 permutation tests, a LOD value of 2.0 was used as the threshold. The mapping results showed that a mature resistance locus, QYr.caas-5BL / QLr.caas-5BL, exists on 5BL, exhibiting resistance to both stripe rust and leaf rust. Figure 1 The flanking markers AX-111031411 and AX-108936241 correspond to a physical region of 663.7-670.1 Mb on the Chinese Spring reference genome (IWGSCRefSeq v1.1), explaining 3.7%-12.3% of the phenotypic variation. The superior alleles originated from Bainong 64. Sequence alignment analysis showed that the flanking marker AX-108936241 is chromosome-specific, therefore it was converted into a KASP marker for marker-assisted selection breeding.

[0082] Example 2: Establishment of a method for obtaining and detecting the KASP-labeled K_AX-108936241 specific primer set.

[0083] 1. Obtaining the KASP-tagged K_AX-108936241 specific primer set

[0084] KASP marker K_AX-108936241 specific primer set design: Sequence 1 is a flanking sequence of SNP AX-108936241, with its 47th base being either A or T, one of the two sequences of this SNP. Sequence 1 was uploaded to the Polymarker website (http: / / www.polymarker.info / ) for primer design, and a chromosome-specific primer set was selected based on the design results. The K_AX-108936241 primer set includes two competing primers (LF1 and LF2) and one universal primer (LR). The 5' ends of the two positively competing primers were modified by adding universal fluorescent FAM and HEX adapter sequences, respectively, based on the Polymarker primer design results.

[0085] Sequence 1: W represents A or T.

[0086] Primer LF1: (Sequence 2, the lowercase part is the FAM fluorescence-matched adapter sequence, and the bold italic letters are polymorphic sites);

[0087] Primer LF2: (Sequence 3, the lowercase part is the HEX fluorescence-matched adapter sequence, and the bold italic letters are polymorphic sites);

[0088] Primer LR: 5'-CCCCTGCATCCATCGTGTAA-3' (sequence 4).

[0089] The single-stranded DNA molecule represented by primer LF1 and the single-stranded DNA molecule represented by primer LR amplify the fragment with genotype AA at the AX-108936241 site.

[0090] The single-stranded DNA molecule represented by primer LF2 and the single-stranded DNA molecule represented by primer LR amplify the TT fragment at the AX-108936241 locus.

[0091] The single-stranded DNA molecules represented by primer LF1, primer LF2, and primer LR amplify the AT fragment at the AX-108936241 locus.

[0092] The genotype of the wheat at the AX-108936241 locus can be obtained by sequencing or by fluorescence development after a KASP reaction.

[0093] 2. Establishment of a detection method for the specific primer set corresponding to the KASP marker K_AX-108936241

[0094] 1) Test materials: Jingshuang 16, Bainong 64 and Jingshuang 16 / Bainong 64 recombinant inbred line populations (including 171 families).

[0095] 2) Extract genomic DNA from the test material and dilute it with ddH2O to obtain a reaction solution with a concentration of 50 ng / μL.

[0096] 3) PCR reaction: Using the diluted DNA reaction solution as a template, PCR amplification was performed using specific primers K_AX-108936241 to obtain the PCR product.

[0097] Preparation of KASP-labeled primer working solution:

[0098] Take 12 μL (100 μM) of upstream primer (primer shown in sequence 2), 12 μL of downstream primer (primer shown in sequence 3), and 30 μL (100 μM) of universal primer (primer shown in sequence 4), respectively, and make up to 100 μL with sterile water. Mix thoroughly and use as working solution for KASP-labeled primers.

[0099] The PCR reaction system is shown in Table 1:

[0100] Table 1. PCR reaction system for K_AX-108936241

[0101]

[0102] PCR amplification program: 95℃ for 15 min; 95℃ for 20 s, 65-57℃ for 1 min (decreasing by 0.8℃ per cycle), 10 cycles; 95℃ for 20 s, 57℃ for 60 s, 32 cycles.

[0103] 4) Genotyping: After the PCR reaction, genotyping was performed using the PHERAstar Plus autofocus fluorescence multi-functional microplate reader (BMG LABTECH) and Klustercaller v3.4 software (LGC, Hoddesdon, UK).

[0104] The results are as follows Figure 2As shown, blue samples represent the AA genotype (Jingshuang 16 type) linked to the FAM fluorescent tag sequence; red samples represent the TT genotype (Bainong 64 type) linked to the HEX fluorescent tag sequence; green samples represent the AT heterozygous genotype; and pink samples may have an undetermined genotype due to poor genomic DNA quality. Comparing the genotyping results of the Jingshuang 16 / Bainong 64RIL population with the original microarray genotyping, except for samples with undetermined genotypes, the marker detection results for other families were consistent with the microarray data, proving that K_AX-108936241 transformation was successful.

[0105] Example 3: Actual Sample Detection

[0106] 1. Test Materials: 322 natural varieties or lines from domestic and international sources; family names are listed in column 2 of Table 2. This set of materials was planted in Tianshui in 2020-2021, and in Tianshui and Yangling in 2021-2022 to obtain stripe rust phenotypes in three environments; it was also planted in Zhengzhou in 2020-2021, and in Baoding in 2021-2022 to obtain leaf rust data in two environments. Under all stripe rust conditions, the inducer variety Mingxian 169 was planted perpendicular to the planting direction of the population materials, and inoculated with stripe rust physiological races CYR32 and CYR34 to promote disease development. Under all leaf rust conditions, the inducer variety Zhengzhou 5389 was planted perpendicular to the planting direction of the population materials, and inoculated with leaf rust physiological races THTT, THJH, PHTH, and FHRL to promote disease development. All field trials used a completely randomized block design with two replicates, single-row plots, row length of 1m, row spacing of 0.25m, 50 seeds per row, and every 17 rows planted the two parents and the susceptible control Mingxian 169 (stripe rust) or Zhengzhou 5389 (leaf rust).

[0107] 2. Phenotypic identification of stripe rust and leaf rust in mature plants: In the mature plant stage, when the stripe rust-susceptible control Mingxian 169 or the leaf rust-susceptible control Zhengzhou 5389 fully develops the disease, a phenotypic survey is conducted, and the ratio of the area of ​​the spore mass on the infected leaf to the total leaf area is recorded for each family.

[0108] 3. The K_AX-108936241 marker detection steps are as follows:

[0109] 1) Genomic DNA was extracted from the above-mentioned test materials using a high-salt, low-pH method, and then diluted to obtain a template solution with a DNA concentration of approximately 50 ng / μL.

[0110] 2) The genotypes of the tested materials were analyzed using the K_AX-108936241 primer set as described in Example 2. The results are as follows: Figure 3As shown, there were 58 samples with the AA genotype (Jingshuang 16 type), 261 samples with the TT genotype (i.e., Jingshuang or Xindong 22 type), and 3 samples with unknown genotypes. The genotype results of the tested materials are listed in column 3 of Table 2.

[0111] Table 2. Mean phenotypic values ​​of stripe rust and leaf rust in 322 natural varieties at mature stage and detection results of molecular marker K_AX-108936241

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] Note: NA represents an unknown genotype, / indicates hybridization, the left side represents the maternal parent, and the right side represents the paternal parent.

[0125] 4) The PROC TTEST model in the internationally used SAS 9.2 statistical software was used to conduct t-tests on the stripe rust and leaf rust phenotypes of 322 tested materials with different genotypes at the adult stage. The results are shown in Table 3. The results show that the stripe rust and leaf rust phenotypes of TT genotype samples were significantly lower than those of AA genotype samples.

[0126] That is, the wheat tested with the K_AX-10893624 SNP site of the TT genotype showed higher resistance to stripe rust and leaf rust than the wheat tested with the AA genotype.

[0127] Table 3. Phenotypic t-tests of stripe rust and leaf rust in 322 natural varieties at mature stage

[0128]

[0129] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A composition for detecting a single nucleotide polymorphism or a genotype of a specific SNP site in a wheat genome, characterized in that, The SNP site is the 47th nucleotide of the DNA fragment shown in SEQ ID NO: 1 in the wheat genome, and the nucleotide species is A or T; the composition comprises competitive primer A, competitive primer B and universal primer, which are respectively shown as follows: A1) the competitive primer A is a single-stranded DNA with a nucleotide sequence shown in SEQ ID NO: 2, the competitive primer B is a single-stranded DNA with a nucleotide sequence shown in SEQ ID NO: 3, and the universal primer is a single-stranded DNA with a nucleotide sequence shown in SEQ ID NO: 4; A2) the competitive primer A is a single-stranded DNA with a nucleotide sequence shown in SEQ ID NO: 2, the competitive primer B is a single-stranded DNA with a nucleotide sequence shown in SEQ ID NO: 3, and the universal primer is a single-stranded DNA with a nucleotide sequence shown in SEQ ID NO:

4.

2. The composition of claim 1, wherein, In the composition, the ratio of the amount of substance of the competitive primer A, the competitive primer B and the universal primer is 2:2:

5.

3. A reagent characterized in that, The reagent is a reagent containing the composition of claim 1 or 2.

4. Kit characterized in that, The kit is a kit containing the composition of claim 1 or 2 or / and the reagent of claim 3.

5. A DNA molecule characterized in that, The DNA molecule is a DNA molecule comprising the SNP site of claim 1.

6. Use of the SNP site of claim 1 or / and the DNA molecule of claim 5 in identifying or assisting in identifying the resistance to wheat stripe rust or / and leaf rust or / and as a molecular marker of the resistance to wheat stripe rust or / and leaf rust.

7. Use, characterized in that, The use is the use of the composition of claim 1 or 2, the reagent of claim 3, the kit of claim 4 or / and the DNA molecule of claim 5 in at least one of the following: B1) use in identifying or assisting in identifying the resistance to wheat stripe rust or / and leaf rust; B2) use in preparing a product for identifying or assisting in identifying the resistance to wheat stripe rust or / and leaf rust; B3) use in comparing or assisting in comparing the resistance to wheat stripe rust or / and leaf rust; B4) use in preparing a product for comparing or assisting in comparing the resistance to wheat stripe rust or / and leaf rust; B5) use in identifying or assisting in identifying an excellent gene for the resistance to wheat stripe rust or / and leaf rust; B6) use in preparing a product for identifying or assisting in identifying an excellent gene for the resistance to wheat stripe rust or / and leaf rust; B7) use in screening, breeding, assisting in screening or assisting in breeding a wheat single plant, strain, line or variety containing an excellent gene for the resistance to wheat stripe rust or / and leaf rust; B8) use in preparing a product for screening, breeding, assisting in screening or assisting in breeding a wheat single plant, strain, line or variety containing an excellent gene for the resistance to wheat stripe rust or / and leaf rust; B9) use in wheat breeding and / or assisting in breeding; B10) use in preparing a product for wheat breeding and / or assisting in breeding.

8. A method for breeding a single plant or a line or a strain or a variety of wheat with resistance to stem rust or / and leaf rust, characterized in that, The method comprises the step of selecting wheat with genotype TT of the SNP site as claimed in claim 1 as a parent for breeding, wherein the TT genotype indicates that the SNP site as claimed in claim 1 is in homozygous T form in the wheat genome.

9. A method for identifying or aiding in the identification of a wheat plant having resistance to stem rust or / and leaf rust, characterized in that, The method comprises the step of identifying or assisting in identifying the resistance to stripe rust or / and leaf rust of wheat according to the polymorphism or genotype of the SNP site as claimed in claim 1 of the wheat to be tested, wherein the wheat to be tested with genotype TT of the SNP site is or is a candidate for wheat with resistance to stripe rust or / and leaf rust. Or / and The wheat to be tested with genotype TT of the SNP site has or is a candidate for higher resistance to stripe rust or / and leaf rust than the wheat to be tested with genotype AA or AT of the SNP site. The TT genotype indicates that the SNP site as claimed in claim 1 is in homozygous T form in the wheat genome; the AA genotype indicates that the SNP site as claimed in claim 1 is in homozygous A form in the wheat genome; and the AT genotype indicates that the SNP site as claimed in claim 1 is in heterozygous form of A and T in the wheat genome.

10. A method for screening and removing wheat plants with relatively lower resistance to stripe rust or / and leaf rust, the method comprising detecting the genotype of the SNP site as claimed in claim 1, and removing the wheat plants with genotype AA or AT of the SNP site, wherein the AA genotype indicates that the SNP site as claimed in claim 1 is in homozygous A form in the wheat genome; and the AT genotype indicates that the SNP site as claimed in claim 1 is in heterozygous form of A and T in the wheat genome.

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