Molecular markers for grain length in wheat and their applications

Abstract

The application discloses a wheat kernel length molecular marker and application thereof, and solves the technical problem of how to provide a wheat kernel length molecular marker. The application specifically discloses the following application of any one of an InDel molecular marker or a substance for detecting the InDel molecular marker: A1) application in identification or auxiliary identification of wheat kernel length; A2) application in preparation of a product for identification or auxiliary identification of wheat kernel length; A3) application in preparation of a wheat breeding product; the InDel molecular marker is a DNA molecule with a nucleotide sequence of sequence 5. The kernel length of the deletion type wheat is longer than or longer than the kernel length of the insertion type or heterozygous type wheat, and can be used in industrial production.

Description

Technical Field

[0001] This invention relates to molecular markers for wheat grain length and their applications in the field of biotechnology. Background Technology

[0002] Wheat is one of the most widely cultivated food crops in the world, and its yield directly impacts global food security. Wheat grain yield is composed of the number of ears per unit area, the number of grains per ear, and grain weight; coordinating these three factors is key to achieving high wheat yields. Grain weight (thousand-grain weight), as one of the yield components, is relatively stable and less affected by environmental conditions, and has always been valued by breeders. Increasing grain weight is an important approach to high-yield wheat breeding.

[0003] Grain weight, as an important component of yield, possesses relatively stable heritability and is composed of multiple sub-components, including grain morphology parameters and grain filling rate. Grain morphology parameters can be further broken down into basic components such as grain length, grain width, and grain thickness. After flowering and fertilization in wheat, the fertilized egg first divides, forming the endosperm nucleus within the ovary, which proliferates along the edge of the embryo sac. Finally, the embryo sac is filled with endosperm cells, and the proliferation of the embryo and endosperm tissues is completed roughly simultaneously. In terms of time, the increase in wheat grain volume occurs first in length, followed by width and thickness. Generally, grain length reaches its maximum value 12-15 days after flowering. After reaching its maximum length, width and thickness increase rapidly, at which point grain development is essentially complete. Afterward, it begins to accumulate nutrients in large quantities, entering the grain filling and ripening stage. Wheat grain weight is influenced by various factors during its formation, including not only genetic factors but also external environmental factors such as light, temperature, and nutrients. Therefore, wheat grain yield is highly susceptible to adverse external factors such as high temperatures, drought, and hot, dry winds after flowering. High temperatures, weak light, and drought during the grain-filling stage can significantly reduce the thousand-grain weight of wheat. From a developmental perspective, the morphological formation of grain length occurs earlier than that of width and thickness, thus it is less affected by later environmental factors, and its trait expression is more stable, which is beneficial for its application in breeding. Summary of the Invention

[0004] The technical problem solved by this invention is how to identify or assist in the identification of wheat grain length.

[0005] To address the aforementioned technical problems, the present invention provides the following applications.

[0006] Applications of InDel molecular labeling or detection of InDel molecularly labeled substances in any of the following ways:

[0007] A1) Application in identifying or assisting in the identification of wheat grain length;

[0008] A2) Applications in the preparation of products for identifying or assisting in the identification of wheat grain length;

[0009] A3) Application in the preparation of wheat breeding products;

[0010] The InDel molecular marker is a DNA molecule with nucleotide sequence Sequence 5.

[0011] In this application, the indicator of seed length can be ten-seed length or single-seed length.

[0012] The substance may be a product. The detection substance may include reagents, kits, and instruments for detecting the above-mentioned InDel molecular markers. Specifically, primers and / or other reagents and instruments required for in vitro nucleic acid amplification for detecting the above-mentioned InDel molecular markers.

[0013] In this application, sequence 5 is position 592704585-622669697 of the sequence with GeneBank number GenBank:CM031193.1(06-MAY-2021).

[0014] To address the aforementioned technical problems, the present invention also provides a product.

[0015] The product is a substance containing the InDel molecular marker described above, and may be any one of the following G1)-G3):

[0016] G1) Products that detect the InDel molecular marker;

[0017] Application of G2 in identifying or assisting in the identification of wheat grain length;

[0018] G3) Application in the preparation, identification, or auxiliary identification of wheat grain length products;

[0019] Application of G4 in the preparation of wheat breeding products.

[0020] To address the aforementioned technical problems, the present invention also provides a method for identifying or assisting in the identification of wheat grain length.

[0021] The method includes detecting wheat genotypes to identify or assist in identifying wheat grain length:

[0022] The grain length of wheat with the deletion genotype is longer than that of wheat with the insertion genotype or heterozygous genotype. The deletion genotype is a homozygous genotype with the InDel molecular marker present. The insertion genotype is a homozygous genotype without the InDel molecular marker present. The heterozygous genotype is a heterozygous genotype with and without the InDel molecular marker present.

[0023] To address the aforementioned technical problems, this invention also provides a method for wheat breeding.

[0024] The method includes selecting homozygous wheat with the aforementioned InDel molecular marker in its genome as parents for breeding.

[0025] The above describes the application of the aforementioned methods in wheat breeding.

[0026] In the above-mentioned applications, products, and methods, the purpose of breeding includes cultivating or selecting wheat with long or short grain lengths.

[0027] In the above applications, products, methods, or procedures, the substance used to detect the InDel molecular marker is one of the following: D1), D2), D3), or D4):

[0028] D1) In vitro nucleic acid amplification primers containing specific InDel molecular markers;

[0029] D2) An in vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primers described in D1);

[0030] D3) A kit containing the in vitro nucleic acid amplification primers described in D1) or the in vitro nucleic acid amplification reagents described in D2);

[0031] D4) A detection instrument containing the in vitro nucleic acid amplification primers described in D1), the in vitro nucleic acid amplification reagents described in D2), or the kit described in D3).

[0032] The in vitro nucleic acid amplification technology may be polymerase chain reaction (PCR), chain substitution amplification (SDA), ligase chain reaction (LCR), sequence-dependent amplification (NASBA), rolling circle nucleic acid amplification (RCA), loop-mediated isothermal amplification (lamp), helicase-dependent isothermal amplification (HDA), or Qβ replication technology.

[0033] This application uses polymerase chain reaction (PCR) as an amplification method to detect polymorphism.

[0034] The specific amplification described in D1) can detect the nucleotide sequence of SNP polymorphic sites by the presence or absence of amplification products or by combining the presence or absence of amplification products with auxiliary reagents such as probes.

[0035] In the above applications, products, or methods, the in vitro nucleic acid amplification primers include primer pair 1 and primer pair 2:

[0036] The primer pair 1 consists of marker 1-F and marker 1-R;

[0037] The primer pair 2 consists of marker 2-F and marker 2-R;

[0038] The nucleotide sequences marked 1-F are single-stranded DNA of SEQ ID No. 2 in the sequence listing;

[0039] Marker 1-R indicates that the nucleotide sequence is the single-stranded DNA of SEQ ID No. 3 in the sequence listing;

[0040] Marker 2-F indicates that the nucleotide sequence is the single-stranded DNA of SEQ ID No. 4 in the sequence listing;

[0041] The nucleotide sequence marked 2-R is the single-stranded DNA of SEQ ID No. 5 in the sequence listing.

[0042] To address the aforementioned technical problems, the present invention also provides a method for wheat breeding.

[0043] The method involves performing a deletion mutation on the sequence 5 region of the target wheat genome to obtain a deletion homozygous wheat, resulting in wheat with grains longer than the target wheat.

[0044] To address the aforementioned technical problems, the present invention also provides the following applications.

[0045] The sequence from GenBank:CM031193.1(06-MAY-2021) or the sequence from position 1 to position 592704584 in GenBank:CM031193.1(06-MAY-2021) corresponds to different grain lengths in any of the following applications:

[0046] A1) Application in identifying or assisting in the identification of wheat grain length;

[0047] A2) Applications in the preparation of products for identifying or assisting in the identification of wheat grain length;

[0048] A3) Application in the preparation of wheat breeding products.

[0049] To address the aforementioned technical problems, the present invention also provides a DNA molecule.

[0050] The DNA molecule is a DNA molecule with nucleotide sequence 592704585-622669697.

[0051] The in vitro nucleic acid amplification technology may be polymerase chain reaction (PCR), chain substitution amplification (SDA), ligase chain reaction (LCR), sequence-dependent amplification (NASBA), rolling circle nucleic acid amplification (RCA), loop-mediated isothermal amplification (lamp), helicase-dependent isothermal amplification (HDA), or Qβ replication technology.

[0052] This application uses polymerase chain reaction (PCR) as an amplification method to detect polymorphism.

[0053] In the above applications, methods, and products, the in vitro nucleic acid amplification primers 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 labels, 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.

[0054] The wheat may be at least one of the following:

[0055] The wheat can be a hybrid offspring of wheat A and wheat B, and the InDel molecular marker is a naturally occurring deletion mutation. Both wheat A and wheat B are insertion types. The hybrid offspring can be F2 generation or higher, such as F2 generation, BC1F2, RIL population, etc.

[0056] The purpose of the breeding program includes developing wheat with longer grains.

[0057] The wheat variety A can be local yellow wheat. The wheat variety B can be Zhengmai 9023.

[0058] In the above applications, methods, and products, the substance may be a reagent and / or kit and / or instrument required to determine the InDel molecular marker or genotype by at least one of the following methods: in vitro nucleic acid amplification, DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and InDel chips. InDel chips include chips based on nucleic acid hybridization reactions, chips based on single-base extension reactions, chips based on allele-specific primer extension reactions, chips based on one-step reactions, chips based on primer ligation reactions, chips based on restriction endonuclease reactions, chips based on protein-DNA binding reactions, and chips based on fluorescent molecule-DNA binding reactions.

[0059] Beneficial effects

[0060] This invention discloses a molecular marker for wheat grain length and its applications, addressing the technical problem of providing a molecular marker for wheat grain length. Specifically, it discloses the application of the InDel molecular marker or a substance for detecting the InDel molecular marker in any of the following ways: A1) application in identifying or assisting in the identification of wheat grain length; A2) application in preparing products for identifying or assisting in the identification of wheat grain length; A3) application in preparing wheat breeding products; wherein the InDel molecular marker is a DNA molecule with a nucleotide sequence of sequence 5. The grain length of deletion-type wheat is longer than that of insertion-type / hybrid wheat, and it can be used for industrial production. Attached Figure Description

[0061] Figure 1 Electrophoresis results of genotype detection of materials GL and Jing 411 using BC3F4 strains 1-8.

[0062] Figure 2 The image shows the phenotypic diagrams of ten grains obtained from the 1-8 genotypes of the GL and Jing 411 materials using the BC3F4 strain.

[0063] Figure 3 The bar chart shows the statistical results of grain length in the BC3F4 line of materials GL and Jing411 with insertion / heterozygous and deletion genotypes. Detailed Implementation

[0064] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and 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 invention in any way.

[0065] 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.

[0066] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0067] 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).

[0068] 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.

[0069] 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.

[0070] The term "InDel molecular marker" refers to InDel markers (short for Insertion and Deletion markers), which are insertion or deletion events occurring in the genome, based on nucleotide insertions or deletions occurring at specific locations within the genome. Similar to alleles, multiple InDel molecular marker genotypes can exist. For example, in diploids, the InDel molecular marker 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 and described based on the specific allele distribution.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] The term "deletion mutation" refers to the process by which a codon encoding a certain amino acid is changed to a codon encoding another amino acid after a base is deleted. "Deletion mutations" include, but are not limited to, single-base deletions, multi-base deletions, base fragment deletions, chromosomal fragment deletions, and chromosomal deletions.

[0075] The term "deletion homozygote" refers to an individual whose chromosomal structural variations include paired deletion mutations in chromosomal segments. Such individuals include, but are not limited to, cells, cell lines, tissues, organs, and intact individuals.

[0076] The term "wheat" refers to an annual or biennial herbaceous plant belonging to the Poaceae family and the Triticum genus.

[0077] The terms “include,” “including,” “have,” “contain,” etc., are all open-ended terms, meaning that they include but are not limited to.

[0078] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0079] The term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecule & Lar Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; and the local homology algorithm of Pearson et al. (1988) P… Similarity search methods are described in roc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see AltschμL et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAS. T-2 (AltschμL et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLASTAltschμL et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, MountainView, California.

[0080] Material description:

[0081] Zhengmai 9023: Xu Weigang. Nationally Approved New Wheat Variety - Zhengmai 9023 [J]. China Agricultural Information, 2009, (07): 34.

[0082] The local yellow wheat is recorded in the following literature: "Chen Yaru, Zhang Qiaofeng, Fu Bisheng, Cai Shibin, Wu Jizhong, Chen Yahua. Analysis of the differences in lead, cadmium and zinc accumulation in wheat microcore germplasm and screening of low accumulation varieties [J]. Journal of Nanjing Agricultural University, 2017, 40(03):393-399.", and the name in this literature is local yellow wheat.

[0083] GL's accession number is CGMCC No.31802.

[0084] Biomaterial name: GL

[0085] Variety name: Wheat

[0086] Latin name: Triticum aestivum

[0087] Number: CGMCC No. 31802

[0088] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0089] Collection institution abbreviation: CGMCC

[0090] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0091] Deposit date: October 12, 2024

[0092] CGMCC Registration Number: CGMCC No. 31802

[0093] Example 1: Discovery of Molecular Markers

[0094] The molecular marker provided in this invention is derived from a material GL (accession number CGMCC No. 31802) resulting from a cross between local yellow wheat and Zhengmai 9023 wheat and a series of backcrosses with Zhengmai 9023. GL exhibits a significant increase in grain length. Molecular marker identification and sequencing revealed that the grain length trait of this material is controlled by a deletion at the terminal portion of the long arm of chromosome 6A.

[0095] As described above, this invention specifically discloses an Indel molecular marker, named GL, with a specific sequence being a deletion at the end of chromosome 6A. The wheat chromosome 6A sequence is the sequence shown in GeneBank No. GenBank:CM031193.1(06-MAY-2021). Sequence 5 is the region from position 592704585 to position 622669697 of the chromosome 6A sequence [GeneBank No. GenBank:CM031193.1(06-MAY-2021)].

[0096] The above molecular markers have three genotypes: deletion, insertion, and heterozygosity.

[0097] Using physical distance as the standard, the specific description is as follows:

[0098] The two homologous chromosomes in the A subgenome of the deletion type (also known as the deletion genotype) have the following characteristics: the DNA sequence from position 592704585 to position 622669697 (the end) in genome 6A is absent.

[0099] In the A subgenome of the insertion type (also known as the insertion genotype), both homologous chromosomes share the following characteristics: the DNA sequence in genome 6A is present from position 592704585 to position 622669697 (the end).

[0100] One homologous chromosome of the A subgenome of the heterozygous type (also known as the heterozygous genotype) has the following characteristics: the DNA sequence from position 592704585 to position 622669697 (terminal) in genome 6A is absent. The other homologous chromosome has the following characteristics: the DNA sequence from position 592704585 to position 622669697 (terminal) in genome 6A is present.

[0101] Using a specific sequence as the standard, the details are as follows:

[0102] The two homologous chromosomes in the A subgenome of the deletion type (also known as the deletion genotype) both have the following characteristics: sequence 5 is absent in the genome.

[0103] Both homologous chromosomes in the A subgenome of the insertion type (also known as the normal genotype) have the following characteristics: sequence 5 is present in the genome.

[0104] One homologous chromosome of the A subgenome of the heterozygous type (also known as the heterozygous genotype) has the following characteristic: sequence 5 is absent in the genome. The other homologous chromosome has the following characteristic: sequence 5 is present in the genome.

[0105] Example 2: Development of Molecular Marker Primers

[0106] Based on the above missing fragment sequence, the following molecular markers were designed as detection primers. Details of the molecular markers are shown in Table 1 below:

[0107] Table 1

[0108]

[0109] Genotyping methods:

[0110] Wheat genome was extracted and analyzed using marker 1-F / marker 1-R and marker 2-F / marker 2-R. Electrophoresis results showed that if the amplification result of marker 1-F / marker 1-R was approximately 697 bp and that of marker 2-F / marker 2-R was approximately 561 bp, the wheat genotype was either insertional or heterozygous. If neither marker 1-F / marker 1-R nor marker 2-F / marker 2-R amplification was observed, the wheat genotype was deletional. Sequencing of the amplified fragment yielded results consistent with the genotype detection method described above. Therefore, the established genotype detection method can be used for practical testing.

[0111] 1. Extraction of wheat genomic DNA

[0112] Genomic DNA was extracted from wheat materials using conventional methods.

[0113] 2. PCR amplification

[0114] 1) PCR reaction system (Novazia, 2×Rapid Taq Plus Master Mix (Dye Plus))

[0115] Genomic DNA (100 ng / μL): 1 μL;

[0116] Primer F (10μM): 1.0μL;

[0117] Primer R (10μM): 1.0μL;

[0118] 2×PCR buffer for KOD FX: 12.5μL;

[0119] ddH2O: 9.5 μL.

[0120] Example 3: Development and Validation of Molecular Marker Function

[0121] To verify the effect of the missing fragment on grain length, we crossed the material (material GL, which was identified as a deletion type by the above genotyping method and genome sequencing) with the wheat variety Jing 411 (identified as an insertion type by genome sequencing), and backcrossed it with Jing 411 three times, and then self-crossed it four times to obtain the BC3F4 population.

[0122] The BC3F4 population, material GL, and Jing 411 were planted at the Changping Farm of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, during the 2022-2023 growing season, and grown under conventional field management conditions. When the wheat reached maturity, grain length was measured (results are shown in Table 2 and...). Figure 3 ( Figure 3Among them, Jing 411 is the wheat variety Jing 411, GL is the material GL, BC3F4 (insertion or deletion type) is the wheat in the BC3F4 population with the insertion or deletion genotype, BC3F4 (deletion type) is the wheat in the BC3F4 population with the deletion genotype, the unit of the vertical coordinate is millimeter (mm)), and, record the phenotype of the length of ten grains (the results are as Figure 2 shown Figure 2 Among them, Jing 411 is the wheat variety Jing 411, GL is the material GL, BC3F4 line 1, BC3F4 line 2, BC3F4 line 3, BC3F4 line 4, BC3F4 line 5, BC3F4 line 6, BC3F4 line 7 and BC3F4 line 8 respectively correspond to the BC3F4 lines 1-8 in the BC3F4 population Figure 1 .

[0123] Grain length detection method:

[0124] After the wheat materials are harvested, in the grains, randomly measure the total length of 10 grains, divide by 10 and convert it into the length of a single grain, with 4 replicates. For materials GL and Jing 411, 40 individual plants are measured respectively, and for BC3F4 lines, 160 lines are measured. The data of grain length analysis are processed by SPSS 11.5 statistical software. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test is used. P < 0.05 (*) indicates significant difference, P < 0.01 (**) indicates extremely significant difference, and P < 0.001 (***) indicates extremely significant difference.

[0125] Length of ten grains: After the wheat materials are harvested, in the grains, randomly measure the total length of 10 grains, with 4 replicates. For materials GL and Jing 411, 40 individual plants are measured respectively, and for BC3F4 lines, 160 lines are measured. The data are processed by SPSS 11.5 statistical software. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test is used. P < 0.05 (*) indicates significant difference, P < 0.0 (**) indicates extremely significant difference, and P < 0.001 (***) indicates extremely significant difference.

[0126] Among them, the lengths of ten grains of GL, Jing 411 and BC3F4 lines 1-8 are as follows: GL (8.6 cm), Jing 411 (7.5 cm), BC3F4 line 1 (7.4 cm), BC3F4 line 2 (7.7 cm), BC3F4 line 3 (7.5 cm), BC3F4 line 4 (7.3 cm), BC3F4 line 5 (8.6 cm), BC3F4 line 6 (8.5 cm), BC'3F4 line 7 (7.9 cm), BC3F4 line 8 (7.4 cm).

[0127] And the genotype detection method in Example 2 was used to detect the genotypes of the above-grown wheat. The results are shown in Table 2 and Figure 1 ( Figure 1 in which, Jing 411 is the wheat variety Jing 411, GL is the material GL, BC3F4 line 1, BC3F4 line 2, BC3F4 line 3, BC3F4 line 4, BC3F4 line 5, BC3F4 line 6, BC3F4 line 7, and BC3F4 line 8 respectively correspond to the BC3F4 lines 1-8 in the Figure 1 BC3F4 population) as shown. The results showed that among the progeny materials of the BC3F4 population, the grain length of the plants with the deletion marker (deletion type) was significantly longer than that of the plants without deletion (insertion type or heterozygous type). This indicates that this deletion fragment can increase the grain length and can be applied to the genetic improvement of wheat grain shape.

[0128] Table 2 Genotypes and Grain Lengths of the BC3F4 Population

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] The above has detailed the present invention. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application. Some basic features can be applied within the scope of the following appended claims.

Claims

1. Any of the following applications of a substance: A1) Application in identifying or assisting in the identification of wheat grain length; A2) Applications in the preparation of products for identifying or assisting in the identification of wheat grain length; A3) Application in the preparation of wheat breeding products; the purpose of the breeding is to cultivate or select wheat with long or short grains; The substance is either D1), D2), D3), or D4. D1) Primers for in vitro nucleic acid amplification; D2) An in vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primers described in D1); D3) A kit containing the in vitro nucleic acid amplification primers described in D1) or the in vitro nucleic acid amplification reagents described in D2); D4) A detection instrument containing the in vitro nucleic acid amplification primers described in D1), the in vitro nucleic acid amplification reagents described in D2), or the kit described in D3); The in vitro nucleic acid amplification primers include primer pair 1 and primer pair 2: The primer pair 1 consists of marker 1-F and marker 1-R; The primer pair 2 consists of marker 2-F and marker 2-R; The nucleotide sequences marked 1-F are the single-stranded DNA of SEQ ID No. 1 in the sequence listing; Marker 1-R indicates that the nucleotide sequence is the single-stranded DNA of SEQ ID No. 2 in the sequence listing; Marker 2-F indicates that the nucleotide sequence is the single-stranded DNA of SEQ ID No. 3 in the sequence listing; Marker 2-R indicates that the nucleotide sequence is the single-stranded DNA of SEQ ID No. 4 in the sequence listing; The wheat variety is a local yellow wheat, Zhengmai 9023, GL, Jing 411 and / or a hybrid offspring of GL and Jing 411.

2. A method for identifying or assisting in the identification of wheat grain length, characterized in that, The method includes detecting the genome of the wheat to be tested using marker 1-F / marker 1-R and marker 2-F / marker 2-R. If the amplification results of marker 1-F / marker 1-R and marker 2-F / marker 2-R are negative, the genotype of the wheat to be tested is deletion type; if the amplification result of marker 1-F / marker 1-R is 697 bp and the amplification result of marker 2-F / marker 2-R is 561 bp, the genotype of the wheat to be tested is insertion type; if the amplification result of marker 1-F / marker 1-R is 697 bp and the amplification result of marker 2-F / marker 2-R is negative, or the amplification result of marker 2-F / marker 2-R is 561 bp and the amplification result of marker 1-F / marker 1-R is negative, the genotype of the wheat to be tested is heterozygous; the grain length of the wheat to be tested with the deletion genotype is longer than or can be longer than the grain length of the wheat to be tested with the insertion genotype or heterozygous type; The nucleotide sequence marked 1-F is the single-stranded DNA of SEQ ID No. 1 in the sequence listing; The nucleotide sequence marked 1-R is the single-stranded DNA of SEQ ID No. 2 in the sequence listing; The nucleotide sequence marked 2-F is the single-stranded DNA of SEQ ID No. 3 in the sequence listing; The nucleotide sequence marked 2-R is the single-stranded DNA of SEQ ID No. 4 in the sequence listing; The wheat to be tested is a local yellow wheat, Zhengmai 9023, GL, Jing 411 and / or a hybrid offspring of GL and Jing 411.

3. A method for wheat breeding, comprising selecting a test wheat with an insertion or deletion genotype as described in claim 2 as a parent for breeding; the purpose of the breeding is to cultivate or select wheat with long or short grain length; the test wheat is a local yellow wheat, Zhengmai 9023, GL, Jing 411 and / or a hybrid offspring of GL and Jing 411.

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