Molecular marker for regulating plant height of wheat TaGA2ox6-2B and application of molecular marker

Abstract

The invention discloses a wheat TaGA2ox6-2B plant height regulation molecular marker and application, and belongs to the technical field of molecular markers. In order to assist in selection of gene resources and molecular markers related to wheat plant height, the invention provides a primer composition for detecting polymorphism or genotype of an SNP site, the SNP site is 113th nucleotide of position 562218647, such as SEQ ID NO: 4, on wheat 2B chromosome, and the type of the nucleotide is A or G; the primer composition can be used for amplifying a wheat genome DNA (Deoxyribose Nucleic Acid) fragment including the SNP site. By applying the KASP molecular marker and the primer composition developed by the invention, agronomic traits related to wheat plant height and yield can be predicted, the cost is saved, the selection efficiency is greatly improved, the breeding process can be accelerated, and a new possibility is provided for efficient screening and cultivation of high-yield wheat varieties.

Description

Technical Field

[0001] This invention relates to the development of high-throughput molecular markers for wheat and their application in marker-assisted breeding, specifically to the molecular marker TaGA2ox6-2B for regulating plant height in wheat and its application, belonging to the field of biotechnology. Background Technology

[0002] Excessive plant height easily leads to lodging in wheat, resulting in reduced yield. Insufficient plant height results in shaded leaves, poor field ventilation, hindering photosynthesis and increasing susceptibility to pests and diseases. Reduced plant height also decreases wheat biomass, negatively impacting grain accumulation during the grain-filling stage. Moderate plant height enhances lodging resistance and helps improve the harvest index, thus ensuring high and stable yields. Currently, most cloned plant height genes in wheat involve gibberellin metabolism or signal transduction. Gibberellin-2-ketoglutarate-dependent oxygenase (GA2oxidase) catalyzes the inactivation of biologically active gibberellin haplotypes (such as GA1, GA3, and GA4), maintaining gibberellin homeostasis. This study analyzed allelic variation and genetic effects of TaGA2ox6-2B, a member of the wheat GA2oxidase-encoding gene (TaGA2ox) family, identifying its superior haplotype—one that reduces plant height without negatively impacting yield—and developed molecular markers suitable for breeding, providing useful genetic resources and molecular tools for wheat genetic improvement. Summary of the Invention

[0003] The technical problem this invention aims to solve is: how to rapidly and accurately screen genes related to plant height, compare or assist in comparing wheat plant heights, screen or assist in screening wheat with a target plant height, and / or breed or assist in breeding wheat with a target plant height. To solve the above technical problem, this invention adopts the following technical solution:

[0004] This invention provides primer compositions for detecting polymorphisms or genotypes at SNP sites, wherein the SNP site may be located at position 562218647 on wheat chromosome 2B (Chinese Spring Reference Genome RefSeq v1.0), such as nucleotide 113 of SEQ ID NO:4, and the nucleotide type is A or G.

[0005] The composition can be used to amplify wheat genomic DNA fragments, including the SNP sites.

[0006] In this invention, 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 whose emission spectra can be inhibited or shifted by 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).

[0007] In this invention, the composition may consist of single-stranded DNA with nucleotide sequence of positions 22-40 of SEQ ID NO:1, single-stranded DNA with nucleotide sequence of positions 22-40 of SEQ ID NO:2, and single-stranded DNA with nucleotide sequence of SEQ ID NO:3.

[0008] In this invention, the composition may consist of the single-stranded DNA shown in SEQ ID NO:1, the single-stranded DNA shown in SEQ ID NO:2, and the single-stranded DNA shown in SEQ ID NO:3.

[0009] In the composition of the present invention, the molar ratio of the single-stranded DNA shown in SEQ ID NO:1, the single-stranded DNA shown in SEQ ID NO:2, and the single-stranded DNA shown in SEQ ID NO:3 is 2:2:5.

[0010] The present invention also provides a reagent containing the above composition.

[0011] The present invention also provides a kit containing the above-described composition and / or the above-described reagents.

[0012] The present invention also provides a DNA molecule, wherein the DNA molecule includes the above-mentioned SNP sites.

[0013] In some embodiments, the DNA molecule may be a DNA molecule with a nucleotide sequence as shown in SEQ ID NO:4.

[0014] This invention also provides the use of the above-described compositions, reagents, and / or kits in at least one of the following:

[0015] A1) Application in identifying or assisting in the identification of wheat plant height;

[0016] A2) Application in the preparation of products for identifying or assisting in the identification of wheat plant height;

[0017] A3) Application in comparing or assisting in comparing the height of wheat plants;

[0018] A4) Application in the preparation of products for comparative or auxiliary comparison of wheat plant height;

[0019] A5) Application in identifying or assisting in the identification of superior genes for wheat plant height;

[0020] A6) Application in the preparation of products for identifying or assisting in the identification of superior genes for wheat plant height;

[0021] A7) Application in screening, breeding, assisting screening or assisting breeding of wheat individual plants, lines, strains or varieties containing superior wheat plant height genes;

[0022] A8) Application in the preparation of products containing wheat single plants, lines, strains or varieties with superior wheat plant height genes for screening, breeding, assisted screening or assisted breeding.

[0023] A9) Applications in wheat breeding and / or assisted breeding;

[0024] A10) Application in the preparation of products for wheat breeding and / or assisted breeding.

[0025] In this invention, the evaluation indicators for wheat breeding may include plant height.

[0026] In this invention, the purpose of wheat breeding may be to obtain wheat with altered plant height, such as obtaining wheat with reduced plant height or / and obtaining wheat with increased plant height.

[0027] The present invention also provides a method for identifying or assisting in the identification of wheat plant height, the method including the step of identifying or assisting in the identification of wheat plant height based on the polymorphism or genotype at locus 562218647 on chromosome 2B of the wheat to be tested;

[0028] The 562218647 site on wheat chromosome 2B is a SNP site in the wheat genome, such as nucleotide 113 of SEQ ID NO:4, whose nucleotide type is A or G.

[0029] The polymorphism or genotype at locus 562218647 on wheat chromosome 2B can be obtained by detecting any of the following substances:

[0030] B1) Primer composition for amplifying wheat genomic DNA fragments including locus 562218647 on wheat chromosome 2B;

[0031] B2) PCR reagents containing the primer composition described in B1);

[0032] B3) A kit containing the primer composition described in B1) or the PCR reagent described in B2).

[0033] The plant height of the tested wheat species homozygous for nucleotide type A at position 562218647 on wheat chromosome 2B, as described in the wheat genome, was significantly lower than that of the tested wheat species homozygous for nucleotide type G at position 562218647 on wheat chromosome 2B, as described in the wheat genome.

[0034] Furthermore, in the method described above, the method for detecting the genotype of the 562218647 locus on chromosome 2B of the wheat to be tested includes using the genomic DNA of the wheat to be identified as a template, performing PCR amplification using the primer composition described above, and obtaining PCR products; determining the genotype of the 562218647 locus on chromosome 2B based on the sequencing results or fluorescence signal of the PCR products.

[0035] Furthermore, the method specifically includes the following operational steps:

[0036] S1) Using wheat genomic DNA as a template and the above primer combination as amplification primers, PCR amplification was performed to obtain PCR amplification products;

[0037] S2) The drought resistance of the wheat to be tested can be determined or assisted by the genotype at the 562218647 locus on chromosome 2B of the PCR amplification product.

[0038] In the above method, step S2) can determine the genotype at locus 562218647 on chromosome 2B based on the sequencing results or fluorescence colorimetric analysis of the PCR product.

[0039] In this invention, the plant height of wheat with the AA genotype at locus 562218647 on chromosome 2B of the wheat genome is lower than or candidate lower than that of wheat with the GG genotype and / or AG genotype at locus 562218647 on chromosome 2B of the wheat genome.

[0040] In this invention, the 5' end of the single-stranded DNA shown in SEQ ID NO:1 and SEQ ID NO:2 is a specific fluorescent tag sequence.

[0041] In this invention, the single-stranded DNAs shown in SEQ ID NO:1 and SEQ ID NO:2 have different specific fluorescent tag sequences, and are combined with fluorescent probes of different luminescent types.

[0042] In one embodiment of this application, the nucleotides 1-21 of the single-stranded DNA shown in SEQ ID NO:1 are FAM-specific fluorescent tag sequences, and the nucleotides 1-21 of the single-stranded DNA shown in SEQ ID NO:2 are HEX-specific fluorescent tag sequences.

[0043] The AA genotype amplification product, combined with FAM fluorescence, is labeled as blue fluorescence; the GG genotype amplification product, combined with HEX fluorescence, is labeled as red fluorescence. That is, the wheat plants whose amplification products are labeled as blue fluorescence are shorter or lower than those of wheat plants whose amplification products are labeled as red fluorescence.

[0044] This invention also provides a method for wheat breeding, the method comprising any of the following:

[0045] C1) Select wheat with genotype AA at locus 562218647 on wheat chromosome 2B as parent for breeding, wherein the AA genotype represents a homozygous wheat genome with nucleotide type A at locus 562218647 on wheat chromosome 2B.

[0046] C2) Select wheat with the genotype GG at the 562218647 locus on wheat chromosome 2B as the parent for breeding. The GG genotype indicates that the nucleotide type at the 562218647 locus on wheat chromosome 2B in the wheat genome is homozygous for G.

[0047] The purpose of breeding in C1) can be to breed wheat with a reduced plant height. The purpose of breeding in C2) can be to breed wheat with an increased plant height.

[0048] The beneficial technical effects achieved by this invention are as follows:

[0049] Genotyping was performed on 160 wheat lines from the Huang-Huai wheat region of my country using the aforementioned TaGA2ox6-2B molecular marker (rs562218647). Genetic effect analysis revealed that, compared with varieties carrying TaGA2ox6-2B_hap2, varieties carrying TaGA2ox6-2B_hap1 had significantly lower plant height. Figure 3 Therefore, TaGA2ox6-2B_hap1 is an excellent allelic variant. However, the frequency of TaGA2ox6-2B_hap1 in natural populations is less than 20%, indicating its great application potential. Attached Figure Description

[0050] Figure 1 The variant sites and major haplotypes of wheat TaGA2ox6-2B are identified.

[0051] Figure 2To use KASP markers to genotype the TaGA2ox6-2B wheat variety in the Huang-Huai wheat region.

[0052] Figure 3 Genetic effects on plant height and yield between two haplotypes, TaGA2ox6-2B, were analyzed. Detailed Implementation

[0053] I. Terminology in this application:

[0054] Examples of resources describing many of the molecular biology-related terms used in this paper 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., Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, Genes IX, Oxford University Press: New York, 2007.

[0055] 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.

[0056] For ease of understanding of this disclosure, several terms and abbreviations used herein are defined as follows:

[0057] When used in a list of two or more items, the term "and / or" means 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.

[0058] 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.

[0059] 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 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-digested 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 tag sites (STS) (Onozaki et al., Euphaitica 1). 38:255 (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.

[0060] 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).

[0061] 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.

[0062] 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.

[0063] The term "template" refers to any nucleic acid molecule that can be used for the amplification described in this invention. Non-natural double-stranded RNA or DNA can be made into double-stranded DNA for use 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.

[0064] 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.

[0065] 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 (SSSR), ligase chain reaction (LCSR), rapid amplification of cDNA ends, PCR and LCSR, 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.

[0066] II. The technical solution provided in this application:

[0067] 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.

[0068] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0069] The natural populations of the Huang-Huai wheat region in the following examples have been described in: Li F, Wen W, Liu J, Zhang Y, Cao S, He Z, Rasheed A, Jin H, Zhang C, Yan J, Zhang P, Wan Y, Xia X. Genetic architecture of grain yield in bread wheat based on genome-wide association studies. BMCPlant Biol. 2019 Apr 29;19(1):168. doi:10.1186 / s12870-019-1781-3. The biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention; it may not be used for any other purpose.

[0070] Unless otherwise specified, all quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Data were processed using Excel statistical software, and experimental results are expressed as mean ± standard deviation. A t-test was used, and **(P < 0.01) indicates a highly significant difference.

[0071] Example 1: Identification of polymorphic sites and haplotypes in the wheat TaGA2ox6-2B gene

[0072] The ID number (TraesCS2B02G396000) of the TaGA2ox6-2B gene, a member of the GA2ox family, was entered into the wheat genome variation database (Wheat-SnpHub, http: / / wheat.cau.edu.cn / Wheat_SnpHub_Portal / ). Resequencing data from 117 modern Chinese wheat varieties and 20 introduced wheat varieties were used to identify the major allelic variations of TaGA2ox6-2B. Polymorphic sites were retrieved from the genomic region from 1.5kb upstream to 500bp downstream of the gene, identifying three variation sites with physical locations on chromosome 2B (RefSeq v1.0) of 562218647, 562219220, and 562219432. These variation sites form two haplotypes (…). Figure 1 In the TaGA2ox6-2B-hap1 haplotype, the bases at the three mutation sites are A, A, and T, respectively; in the TaGA2ox6-2B-hap2 haplotype, the bases at the three mutation sites are G, G, and A, respectively. The three mutation sites are linked.

[0073] Example 2: Development of molecular markers and specific primer sets for differentiating and identifying TaGA2ox6-2B haplotypes

[0074] Kompetitive allele-specific PCR (KASP) molecular markers can select for target traits at the DNA level. They offer accurate genotyping, high throughput, and low cost, making them one of the most suitable molecular marker technologies for assisted breeding. This invention utilizes Polymarker (https: / / www.polymarker.info / ) to design and develop specific KASP primers based on three variant sites (physical locations on chromosome 2B (Chinese Spring Reference Genome RefSeq v1.0) 562218647, 562219220, and 562219432), as shown in Table 3. Primers targeting 562219220 were unsuccessful, while multiple primer pairs were developed based on the variant at 2B:562218647. Ultimately, one primer combination with good genotyping properties was obtained for identifying the wheat TaGA2ox6-2B haplotype.

[0075] Table 3 Primer compositions designed based on three variant sites.

[0076]

[0077]

[0078] The KASP primer composition consists of two competing primers and one universal primer. The following primer composition was designed and screened targeting the physical location 562218647 on chromosome 2B of wheat in the Chinese spring reference genome RefSeq v1.0:

[0079] Competitive primer F1: 5'-gaaggtgaccaagttcatgctGCCACCGTGTCCGCCACAT-3' (SEQ ID NO: 1, the lowercase part is the FAM fluorescent probe specific recognition sequence);

[0080] Competitive primer F2: 5'-gaaggtcggagtcaacggattGCCACCGTGTCCGCCACAC-3' (SEQ ID NO: 2, the lowercase part is the HEX fluorescent probe specific recognition sequence);

[0081] Universal primer R: 5'-CAGGGTAGCAGTGGTAGTTTGT-3' (SEQ ID NO:3).

[0082] The variant sequence detected by KASP marker is the nucleotide at position 113 from the 5' end of SEQ ID NO:4 in the wheat genome (corresponding to the complementary base of the 3' end base of the two competing primers), which is a deoxynucleotide A or G, represented by R in the ST.26 sequence listing.

[0083]

[0084] Genomic DNA was extracted from the wheat to be tested. The nucleotide type at position 562218647 of wheat chromosome 2B (RefSeq v1.0 of Chinese Spring) was detected using the above-mentioned KASP primer combination. The determination can be made by sequencing the PCR product or by the colorimetric results of the KASP reaction system.

[0085] A: Determined by sequencing results of PCR products:

[0086] (1) The nucleotide of the test site is AA, which is the TaGA2ox6-2B-hap1 haplotype.

[0087] (2) The nucleotide of the test site is GG, which is the TaGA2ox6-2B-hap2 haplotype.

[0088] (3) The nucleotides of the test site are A and G, and the nucleotides of the test site are A / G heterozygous.

[0089] B: Judgment is made based on the colorimetric results of the KASP reaction system:

[0090] (1) The wheat to be tested, which showed FAM fluorescence (blue) when combined with the FAM fluorescent probe, was AA, which was the haplotype TaGA2ox6-2B-hap1.

[0091] (2) The wheat to be tested, which showed HEX fluorescence (red) when combined with the HEX fluorescent probe, was identified by the nucleotide GG at the test site, which is the TaGA2ox6-2B-hap2 haplotype.

[0092] (3) The wheat to be tested that simultaneously shows FAM and HEX fluorescence (green) has nucleotides at the test site that are A / G heterozygous.

[0093] Example 3: Application of KASP markers and their specific primer sets in identifying haplotypes of TaGA2ox6-2B in natural wheat populations

[0094] 1. Field phenotypic identification and data analysis of 160 natural wheat populations in the Huang-Huai region

[0095] Agronomic traits of 160 natural populations in six environments were investigated. The target traits included plant height, heading date, thousand-grain weight, number of grains per ear, number of ears, and grain yield (Li F, Wen W, Liu J, Zhang Y, Cao S, He Z, Rasheed A, Jin H, Zhang C, Yan J, Zhang P, Wan Y, Xia X. Genetic architecture of grain yield in bread wheat based on genome-wide association studies. BMC Plant Biol. 2019 Apr29;19(1):168. doi:10.1186 / s12870-019-1781-3.).

[0096] 2. KASP marker detection of genotypes in wheat varieties from the Huang-Huai region

[0097] (1) Genomic DNA was extracted from young leaves of 160 wheat varieties using the CTAB method.

[0098] The quality and concentration of genomic DNA must meet the requirements for PCR, with the following standards: agarose gel electrophoresis showing a single DNA band without obvious diffusion; a Nanodrop 2100 UV spectrophotometer showing an A260 / A280 ratio between 1.8 and 2.0 (indicating no protein contamination in the DNA sample), an A260 / A230 ratio between 1.8 and 2.0 (indicating low salt ion concentration in the DNA sample), and no obvious light absorption at 270 nm (indicating no phenol contamination in the DNA sample); and the concentration of the wheat genomic DNA to be tested is 100 ng / μL.

[0099] (2) Competitive allele-specific PCR (KASP)

[0100] Using wheat genomic DNA as a template, PCR amplification was performed using the KASP primer set synthesized in Example 2 to obtain the amplification products. The reaction system consisted of: 2.0 μL KASP 2×Master Mix (LGC, catalog number: 13448166), 0.048 μL KASP primers (a mixture of 3 primers, with a total concentration of 50 μM, where the molar ratio of two competing primers to one universal primer was 2:2:5), and 2.0 μL template DNA (100 ng / μL). The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using a touch-down program, decreasing by 0.6℃ per cycle) for 1 min, for 10 cycles; 94℃ denaturation for 20 s, 55℃ for 1 min, for another 31 cycles.

[0101] (3) Fluorescence signal acquisition and genotyping analysis

[0102] Fluorescence signals were detected using the PHERAstar Plus autofocus fluorescence multifunction microplate reader (BMG Labtech GmbH, Ortenberg, Germany), and genotyping was performed using KlusterCaller software (LGC, Hoddesdon, UK). Fluorescence values ​​were read by scanning the FAM and HEX beams of the microplate reader when the PCR amplification products cooled to below 40℃ (FAM fluorescent tags were read at excitation wavelengths of 485nm and emission wavelengths of 520nm, and HEX fluorescent tags were read at excitation wavelengths of 528nm and emission wavelengths of 560nm). The genotype was determined based on the fluorescence signal color. The specific determination principle is as follows: if the wheat sample shows a blue fluorescent signal based on the KASP marker, the genotype at position 562218647 on chromosome 2B is homozygous AA; if the wheat sample shows a red fluorescent signal based on the KASP marker, the genotype at position 5622186471276 on chromosome 2B is homozygous GG. Based on the genotyping using the KASP markers described above, the TaGA2ox6-2B gene can be divided into two haplotypes: the wheat sample with a homozygous AA locus at 562218647 on chromosome 2B is designated as haplotype TaGA2ox6-2B-hap1; the wheat sample with a homozygous GG locus at 562218647 on chromosome 2B is designated as haplotype TaGA2ox6-2B-hap2. The haplotype classification of 160 wheat lines is shown in Table 1, and the detailed classification results can be found in [link to table]. Figure 2 .

[0103] 3. Association analysis between wheat TaGA2ox6-2B genotype and plant height and yield

[0104] Phenotypic data of the natural population and TaGA2ox6-2B genotyping results were analyzed using a t-test in Excel to determine the genetic effects of different haplotypes of TaGA2ox6-2B on plant height and yield. The genetic analysis results for plant height and yield traits are shown in Table 2. Experimental results are expressed as mean ± standard deviation.

[0105] Table 1. Haplotypes and phenotypic BLUE values ​​of molecular marker K-6-2B for 160 representative wheat lines in the Huang-Huai wheat region

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] In Table 1, "hap1" is the abbreviation for "TaGA2ox6-2B-hap1", and "hap2" is the abbreviation for "TaGA2ox6-2B-hap2".

[0114] Table 2. Association analysis of agronomic traits related to plant height and yield between two haplotypes of the TaGA2ox6-2B gene.

[0115]

[0116] Note: In Table 1, "hap1" is the abbreviation for "TaGA2ox6-2B-hap1", and "hap2" is the abbreviation for "TaGA2ox6-2B-hap2". "**" indicates a highly significant difference (P < 0.01); "ns" indicates no significant difference.

[0117] In summary, haplotype analysis results show that TaGA2ox6-2B_hap1 stably reduces plant height under various environmental conditions, but has no negative effect on yield-related traits. The conclusions are as follows:

[0118] The plant height of the wheat sample (haplotype TaGA2ox6-2B-hap1) was lower than or lower than that of the wheat sample (haplotype TaGA2ox6-2B-hap2) which was homozygous for AA at locus 562218647 on chromosome 2B in the wheat genome.

[0119] The plant height of the wheat sample with the GG homozygote at locus 562218647 on chromosome 2B (haplotype TaGA2ox6-2B-hap2) was higher than or candidate higher than that of the wheat sample with the AA homozygote at locus 562218647 on chromosome 2B (haplotype TaGA2ox6-2B-hap1).

[0120] 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 SNP site polymorphisms or genotypes, characterized in that, The SNP site is located at position 562218647 on wheat chromosome 2B, such as nucleotide 113 of SEQ ID NO:4, and its nucleotide type is A or G. The composition is used to amplify wheat genomic DNA fragments, including the SNP sites.

2. The composition according to claim 1, characterized in that, The composition comprises a single-stranded DNA with nucleotide sequence of positions 22-40 of SEQ ID NO:1, a single-stranded DNA with nucleotide sequence of positions 22-40 of SEQ ID NO:2, and a single-stranded DNA with nucleotide sequence of SEQ ID NO:

3.

3. The composition according to claim 1 or 2, characterized in that, The composition comprises the single-stranded DNA shown in SEQ ID NO:1, the single-stranded DNA shown in SEQ ID NO:2, and the single-stranded DNA shown in SEQ ID NO:

3.

4. The composition according to claim 3, characterized in that: In the composition, the molar ratio of the single-stranded DNA shown in SEQ ID NO:1, the single-stranded DNA shown in SEQ ID NO:2, and the single-stranded DNA shown in SEQ ID NO:3 is 2:2:

5.

5. A reagent comprising the composition of any one of claims 1 to 4.

6. A kit containing the composition of any one of claims 1 to 4 and / or the reagent of claim 5.

7. A DNA molecule, wherein the DNA molecule comprises the SNP site described in claim 1.

8. Application, characterized in that, The application is the use of the composition of any one of claims 1 to 4, the reagent of claim 5, or / and the kit of claim 6 in at least one of the following: A1) Application in identifying or assisting in the identification of wheat plant height; A2) Application in the preparation of products for identifying or assisting in the identification of wheat plant height; A3) Application in comparing or assisting in comparing the height of wheat plants; A4) Application in the preparation of products for comparative or auxiliary comparison of wheat plant height; A5) Application in identifying or assisting in the identification of superior genes for wheat plant height; A6) Application in the preparation of products for identifying or assisting in the identification of superior genes for wheat plant height; A7) Application in screening, breeding, assisting screening or assisting breeding of wheat individual plants, lines, strains or varieties containing superior wheat plant height genes; A8) Application in the preparation of products containing wheat single plants, lines, strains or varieties with superior wheat plant height genes for screening, breeding, assisted screening or assisted breeding. A9) Applications in wheat breeding and / or assisted breeding; A10) Application in the preparation of products for wheat breeding and / or assisted breeding.

9. A method for identifying or assisting in the identification of wheat plant height, characterized in that, The method includes the steps of identifying or assisting in the identification of wheat plant height based on the polymorphism or genotype at the 562218647 locus on chromosome 2B of the wheat to be tested. The 562218647 site on wheat chromosome 2B is a SNP site in the wheat genome, such as the nucleotide at position 113 of SEQ ID NO:4, and its nucleotide type is A or G. The polymorphism or genotype at locus 562218647 on wheat chromosome 2B was obtained by detecting any of the following substances: B1) Primer composition for amplifying wheat genomic DNA fragments including locus 562218647 on wheat chromosome 2B; B2) PCR reagents containing the primer composition described in B1); B3) A kit containing the primer composition described in B1) or the PCR reagent described in B2).

10. A method for wheat breeding, characterized in that, The method includes any of the following: C1) Select wheat with genotype AA at locus 562218647 on wheat chromosome 2B as described in claim 1 as the parent for breeding, wherein the AA genotype represents a homozygous wheat genome with nucleotide type A at locus 562218647 on wheat chromosome 2B. C2) Select wheat with the genotype GG at the 562218647 locus on the wheat chromosome 2B as described in claim 1 as the parent for breeding, wherein the GG genotype represents a homozygous wheat genome with nucleotides of type G at the 562218647 locus on the wheat chromosome 2B.

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