Application of KASP molecular markers related to wheat grain weight and grain length and primer combination thereof

Abstract

The present invention discloses an application of a SNP molecular marker for identifying or assisting in the identification of wheat grain weight and grain length, and a primer composition thereof. The method comprises detecting the polymorphism or genotype (allele) of a SNP site in the wheat genome to be tested, and identifying or assisting in the identification of wheat grain weight and grain length based on the genotype. The SNP site is a site on wheat chromosome 4A, and its nucleotide type is T or C, which is the 73rd nucleotide of sequence 1 in the sequence table. The method can be used to predict wheat grain weight and grain length, and to carry out wheat breeding. The substance for detecting the polymorphism and genotype of the SNP site can be combined with other substances (such as substances for detecting single nucleotide polymorphisms or genotypes of other molecular markers associated with wheat grain weight and grain length) to prepare and identify wheat high grain weight and grain length products.

Description

Technical Field

[0001] The invention belongs to the field of gene biotechnology, and particularly relates to an application of a KASP molecular marker related to wheat grain weight and grain length and a primer composition thereof. Background Art

[0002] Wheat is the world's third-largest food crop, providing approximately 19% of humanity's energy needs. However, the growing conflict between a growing population and shrinking arable land, coupled with the increasing frequency of extreme weather events such as drought, frost damage, and high temperatures caused by global climate change, poses significant challenges to wheat production. Therefore, increasing wheat yields is of vital strategic importance for ensuring global food and nutrition security.

[0003] Wheat yield is influenced by factors such as grain weight, number of grains per ear, and number of ears per unit area. Grain weight is primarily determined by grain size, which can be further broken down into components such as grain length, width, and thickness. Wheat grain weight plays a decisive role in increasing yield and is also more heritable. Studies have shown that grain length, width, and grain weight are highly significantly positively correlated. Wheat grain length is primarily formed during the early stages of grain development and is less affected by the environment. Therefore, identifying QTLs associated with grain weight and length and developing closely linked molecular markers are crucial for increasing grain length and yield, and represent a key goal of wheat breeding.

[0004] Molecular marker-assisted selection (MAS) is a direct selection method based on genotype, unaffected by external environmental factors. It is a new technology that has rapidly developed in recent years and is widely used in breeding practices. Commonly used molecular markers (such as RFLP, AFLP, DArT, and SSR) have long detection cycles, cumbersome procedures, and high costs, making them difficult to use for large-scale screening of breeding progeny. KASP (Kompetitive Allele-Specific PCR) tagging, or competitive allele-specific PCR, utilizes different fluorescent groups added to the ends of primers to type the target sequence based on the fluorescence signal at the end of the PCR reaction. It can identify specific SNPs (single-base nucleotide polymorphisms) or InDels (insertions / deletions) contained in the target allele. The identification process is efficient, inexpensive, fast, and convenient, and is capable of high-throughput analysis. This has greatly accelerated the progress of molecular marker-assisted selection and holds broad application prospects in crop breeding. Therefore, the development of KASP markers for identifying increased wheat grain weight and length will provide an effective detection method for breeding high-yield wheat varieties, which is of great significance for ensuring high and stable wheat yields and safeguarding national food security. Summary of the Invention

[0005] The problem to be solved by the present invention is how to perform high-throughput identification or auxiliary identification of wheat grain weight and / or grain length.

[0006] In order to solve the above technical problems, the present invention first provides the use of a substance for detecting the polymorphism or genotype of KASP in the wheat genome in any of the following:

[0007] (1) Identify or assist in identifying wheat kernel weight and / or kernel length;

[0008] (2) Wheat breeding;

[0009] (3) preparing products for identifying or assisting in identifying wheat grain weight and / or grain length;

[0010] (4) preparing wheat breeding products;

[0011] The SNP site is a site on wheat chromosome 4A, the nucleotide type of which is T or C, and is the 73rd nucleotide of sequence 1 in the sequence list.

[0012] The wheat variety Chinese Spring genome sequence (IWGSC_RefSeq_v1.0, http: / / 202.194.139.32 / jbrowse-1.12.3-release / ) was used as the reference genome, and the SNP site was located at 492,525,624 bp on wheat chromosome 4A (specifically, position 73 of sequence 1 in the sequence listing).

[0013] The present invention also provides a method for identifying or assisting in identifying wheat grain weight and / or grain length, comprising detecting the genotype of the SNP site in the wheat genome to be tested, and identifying or assisting in identifying the wheat grain weight and / or grain length according to the genotype, wherein the genotype is TT or CC, the TT is a homozygous type in which the SNP site is T, and the CC is a homozygous type in which the SNP site is C.

[0014] As an embodiment, the method for identifying or assisting in identifying wheat grain weight and / or grain length may include the following steps:

[0015] (1) Using the genomic DNA of wheat to be tested as a template, a primer combination is used to perform KASP; the primer combination consists of primer A, primer B, and primer C;

[0016] The primer A is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence list or a single-stranded DNA whose nucleotide sequence is positions 22-43 of sequence 2 in the sequence list;

[0017] The primer B is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence list or a single-stranded DNA whose nucleotide sequence is positions 22-41 of sequence 3 in the sequence list;

[0018] The primer C is a single-stranded DNA molecule whose nucleotide sequence is sequence 4 in the sequence list;

[0019] (2) After completing step (1), performing fluorescence detection to determine the genotype of the SNP of the wheat to be tested;

[0020] (3) Identifying the grain weight and grain length of the wheat to be tested based on the genotype results: the grain weight and / or grain length of the wheat to be tested whose genotype at the SNP site is CC is better than that of the wheat to be tested whose genotype at the SNP site is TT.

[0021] The application of the above method in wheat breeding also falls within the protection scope of the present invention.

[0022] The present invention also provides a wheat breeding method.

[0023] The wheat breeding method provided by the present invention comprises detecting the genotype of the SNP site in the wheat genome, and selecting wheat with a genotype of CC at the SNP site as a parent for breeding, wherein CC is a homozygous type of C at the SNP site.

[0024] As an implementation method, the wheat breeding method may include the following steps:

[0025] (1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using the above primer set;

[0026] (2) After completing step (1), performing fluorescence detection to determine the genotype of the SNP site of the wheat to be tested;

[0027] (3) Select wheat germplasm with CC genotype for wheat breeding with advantages in grain length and weight.

[0028] In the above method, the primer dissolution and preparation method can be as follows: first, dilute the three primers separately with ddH2O to 100mM, and then prepare the primer working solution as follows: primer A 12μL, primer B 12μL, primer C 30μL, ddH2O 46μL, as the KASP-labeled primer working solution, and store at -20°C until use.

[0029] In the above method, the PCR reaction system can be: 1.5 μL of template DNA, 0.0417 μL of primer working solution, 0.75 μL of 2×KASP Master Mix (LGC Company, Lot No. 13426773), and the reaction system is supplemented to 3 μL with sterile ultrapure water.

[0030] In the above method, PCR amplification can be performed on a high-throughput PCR instrument.

[0031] In the above method, the PCR reaction procedure can be:

[0032] Step 1: pre-denaturation at 94°C for 15 min;

[0033] Step 2: denaturation at 94°C for 20 s, annealing for 20 s (the first annealing temperature was 61°C, and the temperature was lowered by 0.6°C each cycle) for a total of 10 cycles; denaturation at 94°C for 20 s, annealing at 55°C for 1 min for a total of 26 cycles;

[0034] Step 3: Extend at 72°C for 3 minutes and store at 4°C.

[0035] In the above method, the method for determining the genotype of the SNP in the wheat to be tested can be: after the PCR reaction is completed, the fluorescence signal is converted into an analyzable numerical value using a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) to read the fluorescence data of the reaction product (the data reading temperature is below 40°C). The fluorescence scanning results are graphically displayed using the R software package, with T base types having FAM fluorescence distributed near the x-axis; C base types having HEX fluorescence distributed near the y-axis; and samples with no detected signal distributed near the origin.

[0036] The present invention also provides a product for detecting the polymorphism or genotype of a SNP site in a wheat genome.

[0037] The product provided by the present invention for detecting the polymorphism or genotype of the SNP site in the wheat genome contains the above-mentioned substance for detecting the polymorphism or genotype of the SNP site in the wheat genome, and the product is any one of:

[0038] C1) Products for detecting single nucleotide polymorphisms or genotypes associated with wheat grain weight and / or grain length;

[0039] C2) Products that identify or assist in identifying wheat kernel weight and / or kernel length;

[0040] C3) Products for wheat breeding.

[0041] In the above applications, methods, and products, the substance may be a reagent and / or instrument required for determining the polymorphism or genotype of the SNP site by at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP chip. Among them, SNP 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.

[0042] Optionally, the substance is the following D1), D2) or D3):

[0043] D1) the substance is a primer composition for amplifying a wheat genomic DNA fragment including the SNP site;

[0044] D2) the substance is a PCR reagent containing the primer combination described in D1);

[0045] D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0046] Optionally, the amplification may be PCR amplification. The primer composition consists of the primer A, the primer B and the primer C.

[0047] D3) The kit may further comprise KASP Master Mix.

[0048] In the above-mentioned applications, methods and products, the primer composition may be labeled or not labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be attached 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 that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The marker can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or alternatively, it can be charge neutral. The marker can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence comprising the marker is detectable. In some embodiments, the nucleic acid is directly detected (e.g., directly reading the sequence) without a marker. For example, the primer composition may be a primer composition composed of a single-stranded DNA having a nucleotide sequence of positions 22-43 of Sequence 2 in the sequence listing, a single-stranded DNA having a nucleotide sequence of positions 22-41 of Sequence 3 in the sequence listing, and a single-stranded DNA having a nucleotide sequence of positions 4 in the sequence listing. The primer composition may also be a primer set consisting of a single-stranded DNA shown in Sequence 2 in the sequence listing, a single-stranded DNA shown in Sequence 3 in the sequence listing, and a single-stranded DNA shown in Sequence 4 in the sequence listing. Sequence 2 in the sequence listing consists of 43 nucleotides, nucleotides 1-21 are a FAM sequence (as a marker), and nucleotides 22-43 are a specific sequence; Sequence 3 in the sequence listing consists of 41 nucleotides, nucleotides 1-21 are a HEX sequence (as a marker), and nucleotides 22-41 are a specific sequence.

[0049] The present invention also provides a DNA molecule, the nucleotide sequence of which is shown as Sequence 1 in the sequence listing.

[0050] The application of the above-mentioned DNA molecules also falls within the scope of protection of the present invention. The application is specifically any of the following:

[0051] (1) Identify or assist in identifying wheat kernel weight and / or kernel length;

[0052] (2) Wheat breeding;

[0053] (3) preparing products for identifying or assisting in identifying wheat grain weight and / or grain length;

[0054] (4) Prepare wheat breeding products.

[0055] Optionally, in the above application, the DNA molecule serves as a detection target.

[0056] The substances for detecting the polymorphism and genotype of the SNP sites can be combined with other substances (such as substances for detecting single nucleotide polymorphism or genotype of other molecular markers related to wheat grain length and yield) to prepare products for identifying wheat varieties of wheat grain weight and / or grain length.

[0057] Herein, the purpose of the breeding may include cultivating high-yielding wheat. The wheat may be a pure line or an inbred line.

[0058] The present invention provides a primer combination and a method for identifying or assisting in the identification of wheat grain weight and length using the primer combination. The method can be used to predict wheat grain weight and length, enabling early screening of wheat to be selected. It can also be used in molecular marker-assisted wheat breeding, and has important application value in discovering wheat germplasm resources with increased grain length and yield, and in breeding wheat varieties with increased grain length and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The KASP primer locations for different alleles of the common wheat grain weight and grain length QTL qTGW4A.1 on chromosome 4A. The boxes indicate the SNPs at physical locations 492, 525, and 624 bp on chromosome 4A (array site AX-111743032). The upstream and downstream primer positions of the KASP marker are double underlined. The sequence in the figure represents the sequence from physical locations 492, 525, 696 bp to 492, 525, and 551 bp on chromosome 4A.

[0060] Figure 2 This is the result of Kasp_qTGW4A.1 marker detection in 309 wheat germplasm materials. DETAILED DESCRIPTION

[0061] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0062] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0063] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0064] The wheat materials in the following examples are well-known varieties, which are respectively recorded in the following references: "Cao Wenxin, Wan Yingxiu, Zhang Qiqi, Li Yan, Zhang Pingzhi. Evolution of cold resistance of main promoted wheat varieties in Huanghuai wheat region. Journal of Wheat Crops, 2015, 35(1): 57-63.", "Geng Xiaoli, Zhang Yueling, Zang Xinshan, Zhao Yue, Zhang Jinbo, You Mingshan, Ni Zhongfu, Yao Yingyin, Xin Mingming, Peng Huiru, Sun Qixin. Evaluation of heat resistance of excellent wheat varieties (lines) in northern winter wheat region and northern Huanghuai area. Journal of Wheat Crops, 2016, 36(2): 172-181.", "Guo Zongzong, Wang Xiang, Wei Li, Bai Ruiying, Cao Yun, Guo Chuang, Yin Jun. Wheat in Huanghuai wheat region Study on the polymorphism distribution of vernalization gene composition. Journal of Henan Agricultural University, 2014, 48(3): 255-262. "Zhang Shuai, Zhang Xilan, Zhang Na, Zhao Minghui, Qiao Wenchen, Sun Lijing, Li Hui, Fu Xiaoyi, He Mingqi, Ji Jun, Li Junming. Genome-wide association analysis of wheat spike-related traits in the northern part of the Huanghuai wheat region. Acta Agriculturae Boreali-Sinica, 2020, 35(Supplement): 31-39. "Zhang Yingjun, Gao Huimin, Li Ziqian, Hu Mengyun, Sun Lijing, Liu Qian, Lv Liangjie, Li Hui. Exploration and tracing of the fusarium head blight resistance gene in Yunzaozhuangyuan germplasm in the northern part of the Huanghuai winter wheat region. Acta Agriculturae Boreali-Sinica, 2022, 35(2): 196-202. "Chen Shulin, Cheng Xiyong, Yu Kang, Chang Xiangnan, Bi Huihui, Xu Haixia, Wang Junsen, Pei Xingxu, Zhang Ziliang, Zhan Kehui. Genome-wide association study of differences in 14 agronomic traits under low- and high-density planting models based on the 660k SNP array for common wheat. Plant Breeding, 2020, 139(2): 272-283. ". Common wheat germplasm is preserved in the Wheat Research Center, Institute of Cereals and Oils Crops, Hebei Academy of Agricultural and Forestry Sciences. The public can obtain this biological material from the applicant. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0065] Example 1. Obtaining the SNP marker chip locus AX-111743032 and KASP marker primer set for the wheat grain weight and grain length QTL qTGW4A.1 interval

[0066] 1. QTL Mapping and Discovery of Microarray Locus AX-111743032

[0067] A 15K SNP array jointly developed by the research groups of Jia Jizeng and He Zhonghu at the Chinese Academy of Agricultural Sciences was used to genotype 309 wheat varieties. Combined with multi-year phenotypic data on grain weight and grain length, genome-wide association analysis was used to locate QTLs for grain weight and grain length. A QTL controlling grain weight and grain length was identified at SNP locus AX-111743032, which was subsequently converted into a KASP marker for use in marker-assisted selection breeding. SNP locus AX-111743032 is located at position 73 of SEQ ID NO: 1, and its nucleotide type is either T or C. In the sequence listing, "y" in SEQ ID NO: 1 represents T or C.

[0068] 2. Obtaining the primer set for KASP marker AX-111743032

[0069] A primer set for detecting the KASP marker (KASP marker Kasp_qTGW4A.1) based on KASP technology was designed. The KASP primer set consists of two upstream primers (primer A and primer B) and one downstream primer (primer C). The specific sequences are shown in Table 1.

[0070] Table 1 KASP marker primer sequences used to identify allelic variation of QTL qTGW4A.1 in common wheat

[0071]

[0072] Primer A has a FAM fluorescent tag sequence (underlined bases) at its 5' end, and primer C amplifies the fragment where the SNP site AX-111743032 is T. The fluorescent signal of the FAM group can be read using a fluorescent signal reader.

[0073] Primer B is a primer with a HEX fluorescent tag sequence (underlined bases) at the 5' end, and primer C amplifies the fragment with SNP site AX-111743032 as C. The fluorescent signal of the HEX group can be read using a fluorescent signal reader.

[0074] Example 2: Establishment of a method for detecting the genotype of SNP marker AX-111743032 using KASP markers

[0075] The KASP marker Kasp_qTGW4A.1 was used to detect different allele types of wheat grain weight and grain length QTL qTGW4A.1 at physical position 492,525,624 (SNP site AX-111743032) on chromosome 4A.

[0076] 1. PCR amplification system and procedure

[0077] Genomic DNA was extracted from common wheat leaves using the CTAB method and dissolved in 400 μL of TE. DNA quality was assessed by electrophoresis on a 1% agarose gel, ensuring the absence of significant impurities, clear bands, and no degradation. After DNA concentration was measured, the DNA was diluted to a uniform concentration of 28.3 ng / μL. PCR amplification was performed using the diluted wheat genomic DNA as a template.

[0078] Primer sequences are shown in Table 1. Preparation of KASP-labeled primer working solution: Dilute each of the three primers to 100 mM in ddH2O. Then prepare the primer working solution as follows: 12 μL of primer A, 12 μL of primer B, 30 μL of primer C, and 46 μL of ddH2O. Store at -20°C until needed.

[0079] The PCR amplification system was as follows: 1.5 μL of template DNA, 0.0417 μL of primer working solution, 0.75 μL of 2×KASP Master Mix (LGC Company, Lot No. 13426773), and the reaction system was supplemented to 3 μL with sterile ultrapure water.

[0080] The PCR reaction program was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing for 20 s (the first annealing temperature was 61°C, and the temperature was lowered by 0.6°C each cycle) for a total of 10 cycles; denaturation at 94°C for 20 s, annealing at 55°C for 1 min for a total of 26 cycles; extension at 72°C for 3 min, and storage at 4°C.

[0081] A blank control (NTC) in which no template DNA was added to the reaction system was also set up in the experiment, with one control set for each plate.

[0082] 2. Genotyping

[0083] After the PCR reaction is completed, a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) are used to convert the fluorescence signal into analyzable values ​​and read the fluorescence data of the reaction products. Fluorescence scanning results are displayed graphically using the R software package. T base types with FAM fluorescence are distributed near the x-axis; C base types with HEX fluorescence are distributed near the y-axis; samples with no detectable signal are distributed near the origin.

[0084] The excitation wavelength for FAM is 485 nm, and the emission wavelength is 520 nm. The excitation wavelength for HEX is 535 nm, and the emission wavelength is 556 nm. The excitation wavelength for the system reference fluorescence, ROX, is 575 nm, and the emission wavelength is 610 nm.

[0085] The results are as follows Figure 2As shown, if only the fluorescence signal of the HEX group is displayed, the genotype of AX-111743032 of the wheat to be tested is CC (i.e., the SNP site AX-111743032 in the wheat genome is a homozygous type of C); if only the fluorescence signal of the FAM group is displayed, the genotype of AX-111743032 of the wheat to be tested is TT (i.e., the SNP site AX-111743032 in the wheat genome is a homozygous type of T).

[0086] Example 3: Application of KASP marker AX-111743032 in assisting identification of wheat grain weight and length in breeding

[0087] Grain weight and grain length phenotyping of 1.309 wheat accessions

[0088] 309 wheat germplasm materials were sown in 2018-2019, 2019-2020, and 2020-2021 at the Dishang Experimental Station of the Institute of Cereals and Oils Crops, Hebei Academy of Agricultural and Forestry Sciences, Gaocheng District, Shijiazhuang City, Hebei Province. A completely randomized block design was used with a row length of 3m, a row spacing of 22cm, and a plant spacing of 3.6cm. Sowing was done in full moisture, and watering was done once during the jointing stage and the filling stage, with an irrigation rate of 50m 3 After the seeds were harvested, the seed length and weight were measured using a Wanshen SC-G automatic seed analyzer and 1000-grain weight analyzer (Wanshen Testing Technology). The two-tailed t-test was used to statistically analyze the seed length and weight of each replicate.

[0089] 2. Identification of the Genotype of Wheat SNP Marker AX-111743032 Using KASP Markers

[0090] According to the method described in Example 2, genomic DNA of each experimental material was extracted, and the genotype of the tested wheat was detected using the above molecular markers. The results are shown in Table 2 and Figure 2 . Figure 2 In the figure, CC is the wheat material with the genotype of SNP site AX-111743032 being CC, TT is the wheat material with the genotype of SNP site AX-111743032 being TT, and CK is the blank control in which no template DNA is added to the reaction system.

[0091] KASP marker detection showed that 147 of the 309 Chinese wheat germplasms were of the qTGW4A.1a allele type TT (i.e., the genotype of the SNP site AX-111743032 was TT), and 162 germplasms were of the qTGW4A.1b allele type CC (i.e., the genotype of the SNP site AX-111743032 was CC).

[0092] Table 2 Wheat germplasm Kasp_qTGW4A.1 marker detection results and thousand-grain weight data

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] Note: NA represents missing data. Wheat varieties with missing data are not included in the grain weight and grain length statistics.

[0102] Table 3 Wheat germplasm Kasp_qTGW4A.1 marker detection results and grain length data

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] Note: NA represents missing data. Wheat varieties with missing data are not included in the grain weight and grain length statistics.

[0113] The statistical analysis results in Tables 4 and 5 showed that the mean weight and grain length of wheat germplasm with the genotype of SNP site AX-111743032 TT were lower than those of wheat germplasm with the genotype of SNP site AX-111743032 CC in different years, and the two were significantly or extremely significantly different (P<0.05 or P<0.01).

[0114] In wheat breeding for high grain weight and grain length, it is best to select wheat with the CC genotype at the SNP site AX-111743032 as the parent for breeding.

[0115] Table 4 Statistical analysis results of the relationship between allele variation types and 1000-grain weight of QTL qTGW4A.1 in common wheat

[0116]

[0117] Note: Statistical analysis was performed using a two-tailed t-test (P<0.05 indicated a significant difference; P<0.01 indicated an extremely significant difference).

[0118] Table 5 Statistical analysis results of the relationship between allele variation types and grain length of QTL qTGW4A.1 in common wheat

[0119]

[0120] Note: Statistical analysis was performed using a two-tailed t-test (P<0.01 indicated that the difference reached an extremely significant level).

[0121] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims. SEQUENCE LISTING <110> Institute of Cereals and Oils Crops, Hebei Academy of Agricultural and Forestry Sciences <120> Application of KASP molecular markers related to wheat grain weight and grain length and primer combination thereof <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 146 <212> DNA <213> 1 <400> 1 ttttgtcggg gaggaaaatc atcgatcctc atcttccaaa ttgggcactc actattagag 60 ctggaggcgc gaytgctggg tgccgggcta gggtgacatg agggatgcta ggtgctgggc 120 ggggcagtgc tgatgggccc aaccgt 146 <210> 2 <211> 43 <212> DNA <213> 1 <400> 2 gaaggtgacc aagttcatgc tctattagag ctggaggcgc gat 43 <210> 3 <211> 41 <212> DNA <213> 1 <400> 3 gaaggtcgga gtcaacggat tattagagct ggaggcgcga c 41 <210> 4 <211> 24 <212> DNA <213> 1 <400> 4 cctagcatcc ctcatgtcac cct 24

Claims

1. Use of a substance for detecting polymorphism or genotype of a SNP site in a wheat genome in any of the following: (1) Identify or assist in identifying wheat kernel weight and / or kernel length; (2) Wheat breeding; (3) Preparation of products for identifying or assisting in identifying wheat kernel weight and / or kernel length; (4) Preparation of wheat breeding products; The SNP site is a site on wheat chromosome 4A, the nucleotide type of which is T or C, and is the 73rd nucleotide of sequence 1 in the sequence list; The genotype of the SNP site is TT or CC, wherein CC is the homozygous type of the SNP site C, and TT is the homozygous type of the SNP site T; the grain weight and / or grain length of the wheat to be tested whose genotype of the SNP site is CC is higher than that of the wheat to be tested whose genotype of the SNP site is TT; The purpose of the breeding is to select wheat with high grain weight and / or grain length as a parent for breeding.

2. The use according to claim 1, characterized in that: The substance is as follows D1), D2) or D3): D1) the substance is a primer composition for amplifying a wheat genomic DNA fragment including the SNP site; D2) the substance is a PCR reagent containing the primer combination described in D1); D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

3. The use according to claim 2, characterized in that: The primer composition consists of primer A, primer B and primer C; The primer A is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence list or a single-stranded DNA whose nucleotide sequence is positions 22-43 of sequence 2 in the sequence list; The primer B is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence list or a single-stranded DNA whose nucleotide sequence is positions 22-41 of sequence 3 in the sequence list; The nucleotide sequence of primer C is a single-stranded DNA molecule of sequence 4 in the sequence table.

4. A method for identifying or assisting in identifying wheat grain weight and / or grain length, characterized in that: The method includes detecting the genotype of a SNP site in the genome of wheat to be tested, and identifying or assisting in identifying the grain weight and / or grain length of wheat according to the genotype, wherein the SNP site is a site on wheat chromosome 4A, the nucleotide type of which is T or C, and is the 73rd nucleotide of sequence 1 in the sequence table, and the genotype of the SNP site is TT or CC, wherein CC is the homozygous type of the SNP site being C, and TT is the homozygous type of the SNP site being T; the grain weight and / or grain length of the wheat to be tested whose genotype of the SNP site is CC is higher than that of the wheat to be tested whose genotype of the SNP site is TT.

5. Use of the method according to claim 4 in wheat breeding, wherein the purpose of the breeding is to select wheat with high grain weight and / or grain length as a parent for breeding.

6. A method for wheat breeding, characterized in that: The method comprises detecting the genotype of the SNP site described in claim 1 in the wheat genome, selecting wheat with a genotype of CC at the SNP site as a parent for breeding, wherein the CC is a homozygous type of the SNP site being C, and the purpose of the breeding is to select wheat with high grain weight and / or grain length as a parent for breeding.

7. The use of a DNA molecule whose nucleotide sequence is Sequence 1 in the sequence listing in any of the following applications: (1) Identify or assist in identifying wheat kernel weight and / or kernel length; (2) Wheat breeding; (3) Preparation of products for identifying or assisting in identifying wheat kernel weight and / or kernel length; (4) Preparation of wheat breeding products; The 73rd position of sequence 1 in the sequence list is a SNP site, the nucleotide type of the SNP site is T or C, the genotype of the SNP site is TT or CC, the CC is the homozygous type of the SNP site is C, and the TT is the homozygous type of the SNP site is T; the grain weight and / or grain length of the wheat to be tested with the genotype of the SNP site being CC is higher than that of the wheat to be tested with the genotype of the SNP site being TT; The purpose of the breeding is to select wheat with high grain weight and / or grain length as a parent for breeding.

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