KASP primer set related to wheat grain weight qtl site qtgw_2b and application thereof

Abstract

This invention discloses a set of QTL loci associated with wheat grain weight. QTgw_2B Related KASP primer sets and their applications QTgw_2B Located on wheat chromosome 2B, the physical region on the Chinese spring reference genome is 716.55–746.10 Mb. The KASP molecular marker primer set includes primers with nucleotide sequences as shown in SEQ ID NO.3–SEQ ID NO.5, which can achieve… QTgw_2B Rapid and accurate detection of superior allelic variations at loci, simple detection, intuitive results, accurate genotyping and suitable for high-throughput analysis. It can be used for early screening, assisted selection and evaluation of breeding potential of high grain weight genotypes in large-scale breeding materials, providing technical support for wheat grain weight improvement and high-yield molecular breeding.

Description

Technical Field

[0001] This invention relates to wheat genetics and breeding, specifically to a set of QTL loci associated with wheat grain weight. QTgw_2B Related KASP molecular marker primer sets and their applications. Background Technology

[0002] Marker-assisted selection (MAS) breeding is an important breeding method that has emerged with the development of modern molecular biology techniques. It can rapidly and accurately analyze the genetic composition of individuals at the molecular level, enabling direct selection of genotypes for target traits. This greatly overcomes many problems faced in traditional breeding, such as difficulty in trait prediction, large workload, and long cycle, and is an effective way to improve the efficiency of crop breeding (Wang Yujie, Leng Chunxu, Sun Zhongyi, et al. (2022) A brief analysis of the application of biotechnology in crop breeding. Agricultural Science and Technology Communications, 2:4-6). Molecular markers are the core tool for MAS breeding, but traditional molecular markers such as RFLP, AFLP, SSR, and dCAPS generally have shortcomings such as cumbersome detection steps, high cost, and long cycle, making it difficult to meet the needs of high-throughput and precise selection of large-scale genetic materials in the breeding process.

[0003] KASP (Kompetitive Allele-Specific PCR) markers, or competitive allele-specific PCR, are a highly efficient genotyping technique developed in recent years. This technique, based on allele-specific primers and fluorescence signal detection, achieves bicelestemization of specific SNPs and insertion-deletion (InDels) sites by reading and interpreting the fluorescence signal at the PCR terminal. KASP markers offer advantages such as high accuracy, low cost, ease of operation, and high throughput, making them particularly suitable for rapid detection in large-scale breeding populations. This aligns with the demands of modern molecular breeding for efficient, precise, and automated detection, effectively promoting targeted improvement of crop varieties (Kaur B, et al. (2020) Utilization of KASP technology for wheat improvement. Cereal Res Commun, 48:409-421).

[0004] Wheat is one of the world's three major food crops and one of my country's most important staple crops, contributing up to 130 million tons to my country's grain production annually (http: / / www.fao.org / worldfoodsituation / csdb / en / ). Developing high-yield wheat varieties and continuously increasing wheat production are important tasks for ensuring my country's food security and social harmony and stability (Liu Wei, Li Shengnan, Ruan Shuang, et al. (2023) Major Progress and Prospects in Wheat Breeding in my country. Barley and Cereal Science, 40(02):1-6). Wheat yield is composed of the number of spikes per unit area, the number of grains per spike, and the grain weight. There are certain mutual constraints and compensatory relationships among these yield components. Grain weight, usually expressed as thousand-grain weight, is a crucial factor affecting wheat yield. Existing studies have shown that grain weight is closely related to wheat yield improvement and is one of the key traits that need to be focused on in modern high-yield wheat breeding (Qin X, et al. (2015) Wheat yield improvements in China: Pasttrends and future directions. Field Crops Res, 177:117-124; Dai Baosheng, Xing Wei, Zhang Huachong et al. (2021) Correlation analysis and path analysis of quantitative traits and yield traits of wheat varieties in the middle and lower reaches of the Yangtze River. Tillage and Cultivation, 41(05):52-54). Therefore, identifying grain weight-related QTLs / genes, discovering superior allelic variations, and developing efficient molecular markers that can be used for breeding practices are of great significance for improving wheat grain weight and promoting the molecular breeding process of high-yield wheat.

[0005] However, compared with other crops, the molecular improvement of wheat grain weight still faces many difficulties, mainly: (1) Common wheat is an allohexaploid with a large and complex genome, making it difficult to analyze the genetic characteristics of complex quantitative traits such as grain weight; (2) Wheat grain weight is regulated by multiple genes, and most of the reported grain weight-related QTLs have small effects, insufficient stability and scattered distribution, resulting in low utilization rate in breeding; (3) Most grain weight-related QTLs and molecular markers are derived from conventional varieties or mutant materials, and their donor parents have limited comprehensive agronomic trait advantages, which further restricts the application of related loci in high-yield breeding.

[0006] Core parents are important germplasm resources in wheat breeding, typically characterized by rich genetic base, low linkage redundancy, and high general combining ability. The superior loci and chromosomal segments they carry can be stably transmitted in derived offspring and play a crucial role in wheat variety improvement. Therefore, using core parents as research materials, combined with linkage analysis and map-based cloning methods, to deeply explore QTLs / genes related to wheat grain weight formation, and to develop accurate, efficient, and practical molecular markers, is of significant practical importance for promoting the transfer and breeding utilization of grain weight-enhancing loci / genes, and accelerating the selection of high-yielding wheat varieties with excellent comprehensive traits. Summary of the Invention

[0007] To address the aforementioned issues, this invention utilizes a high-density genetic linkage map to detect, for the first time, a grain weight-enhancing locus in Yangmai 158, a key parent wheat variety from the middle and lower reaches of the Yangtze River. QTgw_2B Furthermore, a KASP molecular marker primer set was developed based on flanking SNP markers closely linked to this site for efficient detection and breeding utilization of superior wheat grain weight-related genotypes.

[0008] Specifically, the present invention provides a set of QTL loci related to wheat grain weight. QTgw_2B The relevant KASP molecular marker primer set, which is based on wheat grain weight-enhancing QTL sites. QTgw_2B Developed and obtained. QTgw_2B Located on wheat chromosome 2B, the physical region on the Chinese spring reference genome IWGSC RefSeq v1.0 is 716.55–746.10 Mb, and its flanking SNP molecular marker is 2B_746101501. The KASP molecular marker primer set includes the forward competitive primer KASP-2B_746101501-1 (nucleotide sequence shown in SEQ ID NO.3), the forward competitive primer KASP-2B_746101501-2 (nucleotide sequence shown in SEQ ID NO.4), and the reverse universal primer KASP-2B_746101501-3 (nucleotide sequence shown in SEQ ID NO.5). Different fluorescent tags are attached to the 5′ ends of the aforementioned forward competitive primers KASP-2B_746101501-1 and KASP-2B_746101501-2. Specifically, the 5′ end of KASP-2B_746101501-1 is connected to the FAM fluorescent tag shown in SEQ ID NO.1, and the 5′ end of KASP-2B_746101501-2 is connected to the HEX fluorescent tag shown in SEQ ID NO.2.

[0009] The second aspect of this invention provides the application of the above-described KASP molecular marker primer set in any of the following: (1) Detection of wheat grain weight enhancement sitesQTgw_2B and the location of its related genes; (2) To detect or assist in the detection of wheat grain weight; (3) Breeding and creating high-grain-weight wheat varieties; (4) Sites that enhance wheat grain weight QTgw_2B Polymerization breeding with other desirable trait sites.

[0010] A third aspect of the present invention provides a method for detecting the weight of wheat grains, comprising the following steps: (1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using the above-mentioned KASP molecular marker primer set to obtain the amplification product; (2) Fluorescence signal scanning and genotype clustering analysis were performed on the amplification products to determine the characteristics of the wheat material to be tested. QTgw_ 2B Genotype at the locus; (3) Determine the thousand-grain weight of the wheat material to be tested based on the genotype test results.

[0011] Specifically, in step (1), the total volume of the PCR amplification system is 10 μL, including: 2.5 μL of template DNA with a concentration of 120 ng / μL, 5 μL of 2×KASP Master Mix, 0.14 μL of KASP Assay Mix and 2.36 μL of ddH2O.

[0012] The KASP Assay Mix is ​​prepared as follows: Each 100 μL KASP Assay Mix contains 12 μL of 100 μM positive competitive primer KASP-2B_746101501-1, 12 μL of 100 μM positive competitive primer KASP-2B_746101501-2, and 30 μL of 100 μM reverse universal primer KASP-2B_746101501-3, and 46 μL of ddH2O is added to bring the total volume to 100 μL.

[0013] The PCR amplification program was as follows: 94℃ heat activation for 15 min; 94℃ denaturation for 20 s, annealing and extension at 61-55℃ for 60 s, for a total of 10 touch-down cycles, with the temperature decreasing by 0.6℃ per cycle; 94℃ denaturation for 20 s, 55℃ annealing and extension for 60 s, for a total of 26 cycles; the amplified products were incubated at 30℃.

[0014] Specifically, in step (2), it is determined that the wheat material to be tested... QTgw_2BThe method for determining the genotype at a locus is as follows: when the sample to be tested clusters near the Y-axis in the genotyping clustering diagram and displays a blue FAM fluorescence signal, the sample to be tested is determined to be of the AA genotype; when the sample to be tested clusters near the X-axis in the genotyping clustering diagram and displays a red HEX fluorescence signal, the sample to be tested is determined to be of the GG genotype.

[0015] Specifically, in step (3), the method for judging the weight of a thousand grains of wheat is: the weight of a thousand grains of wheat material carrying the AA genotype is higher than that of wheat material carrying the GG genotype.

[0016] Through the above technical solution, the present invention achieves the following beneficial effects: 1. This invention uses Ningmai 9 and Yangmai 158, the backbone parents of wheat in the middle and lower reaches of the Yangtze River, as materials. Through the construction of high-generation recombinant inbred line populations, multi-year grain weight phenotypic identification, and QTL linkage analysis, a new grain weight-enhancing QTL locus was identified. QTgw_2B The synergistic allelic variation originates from Yangmai 158. This locus can provide a new functional site and excellent gene resource for high-yield wheat breeding, and can be combined with molecular marker-assisted selection to achieve breeding transformation and utilization, thus having high application value.

[0017] 2. This invention is aimed at QTgw_2B A KASP molecular marker closely linked to the site was developed, which enables... QTgw_2B Rapid detection and accurate typing of superior allelic variations are characterized by simple operation, intuitive results, stable typing, and suitability for high-throughput analysis, which can meet the needs of large-scale breeding material superior genotype screening.

[0018] 3. The application method of the KASP molecular marker primer set provided by this invention can be used for early identification and assisted selection of high grain weight genotypes in wheat, improving the targeting and efficiency of breeding selection; it can also assist in... QTgw_2B By combining loci with other desirable trait loci in breeding, we can accelerate the selection and breeding of new wheat varieties with high yield and excellent comprehensive traits. Attached Figure Description

[0019] Figure 1 QTL sites for wheat grain weight enhancement QTgw_2B A schematic diagram of genetic mapping; Figure 2 The graphs show the genotyping effect of the KASP molecular marker combination provided by this invention and the genotyping results of the verification materials; where a is the genotyping effect of the KASP molecular marker combination in the test sample, and b is the detection result of the KASP molecular marker combination in the wheat breeding line used for verification; the coordinate axis values ​​represent the allele fluorescence signal intensity, the black dots represent the negative control (NTC, i.e., the sample without added DNA), and the red and blue dots represent different genotypes respectively. Figure 3The image shows a comparison of grain weight between two different genotypes of the tested wheat material, as detected by KASP molecular markers; AA and GG represent the two genotypes, and TGW represents the thousand-grain weight. Detailed Implementation

[0020] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0021] Unless otherwise specified, the terminology used in this invention generally has the meanings commonly understood by those skilled in the art. Various processes and methods not described in detail in the embodiments are conventional methods well-known in the art.

[0022] Unless otherwise specified, the reagents, equipment, etc. used in the following examples are all commercially available.

[0023] The wheat materials used in this invention are all germplasm resources preserved in the Jiangsu Provincial Crop Germplasm Resource Bank (Crop, Yangzhou University Bank), which can be obtained and used by technical and research personnel in this field.

[0024] Example 1: QTL loci for enhancing wheat grain weight QTgw_2B and obtaining the marked interval This embodiment utilizes a high-generation recombinant inbred line population containing 282 lines, constructed by hybridization of Ningmai 9 (a key wheat parent from the middle and lower reaches of the Yangtze River) as the female parent and Yangmai 158 as the male parent. (Published in 2021) For three consecutive years from 2024 to 2025, the aforementioned recombinant inbred population was planted at the Yangzhou University experimental base (119.40°E, 32.34°N) using single-row sowing with a row length of 1.2 m and a row spacing of 0.3 m, with three replicates. The soil conditions at the experimental base were uniform. Before harvest, consistent field management was provided according to the methods published by Ma Hongxiang et al. (2021) (Ma Hongxiang, Gu Kejun, Chen Huaigu (2021), *100 Questions on Key Practical Technologies for Wheat Industry*, China Agriculture Press). After grain maturity, five plants of uniform growth were randomly harvested from each line. After threshing and natural drying, 1000 seeds were randomly selected for grain weight measurement. The weight was expressed as 1000-grain weight, and the average value was used for subsequent data analysis.

[0025] The whole genome of the above-mentioned recombinant inbred line population was analyzed using an Illumina 90K SNP microarray. Subsequently, the marker data were filtered and redundancy-removed using IciMapping v4.1 software, and a linkage genetic map was constructed and corrected using JoinMap v4.0 software. The final genetic map covered all 21 wheat chromosomes, with a total length of 3022 cM.

[0026] Using combined genotypic and grain weight phenotypic data, QTL mapping analysis was performed on the grain weight trait using QTL IciMapping v4.1 software (Meng L, Li H, Zhang L, et al. (2015) QTL IciMapping: Integrated software for genetic linkage map construction and quantitative trait locus mapping in biparental populations. Crop J 3:269-283). Complete Interval Mapping (ICIM-ADD) was employed for linkage mapping, with a LOD value of 2.5 used as the significance threshold. Ultimately, a stable grain weight-enhancing QTL originating from Yangmai 158 and detectable under different environmental conditions was identified, which the applicant named [QTL name missing]. QTgw_2B The genetic mapping results are as follows: Figure 1 As shown. QTgw_2B Located on wheat chromosome 2B, the SNP molecular markers on both sides are 2B_716553672 and 2B_746101501, which correspond to the physical region of 716.55-746.10 Mb on the Chinese spring reference genome IWGSC RefSeq v1.0.

[0027] Example 2: Development, evaluation, screening, and genotyping efficacy verification of KASP molecular markers To better understand the particle weight enhancement sites identified in Example 1 QTgw_2B Based on breeding and utilization, QTgw_ 2B Based on the composition of tightly linked SNP markers, KASP molecular markers suitable for high-throughput genotyping detection were further developed. As shown in Table 1, this invention selected SNP marker 2B_746101501 as the target site for KASP primer development.

[0028] First, 150 bp DNA sequences were extracted upstream and downstream of the SNP marker 2B_746101501. Based on the base composition of the target SNP site, candidate primers were designed using Primer Premier 5 software. The designed candidate primers included two forward primers that specifically bind to the target site and one common reverse primer.

[0029] Subsequently, the quality of candidate primers was evaluated using DNAMAN software. To ensure primer amplification efficiency, detection sensitivity, and genotyping resolution, candidate primers underwent further manual screening. Screening principles included: no consecutive 5 or more complementary base sequences within a single primer, and no consecutive 4 or more complementary base sequences at the 3′ end of the primer that are complementary to other sequences within the primer; no consecutive 6 or more complementary base sequences between the forward and reverse primers; primer Tm values ​​controlled between 59℃ and 65℃; primer length not less than 19 bp, and amplified fragment length not exceeding 150 bp.

[0030] Given that wheat is an allohexaploid crop with numerous homologous and repetitive sequences, the same fragment may exist in multiple copies throughout the genome. To avoid primer mismatch and non-specific amplification, the primer sequences were further aligned using the Ensembl Plants database to confirm their ability to specifically identify the target site. Ultimately, three core primers that met the above design principles and could specifically detect the target site were selected, including two forward primers and one reverse primer.

[0031] Furthermore, different fluorescent tag sequences were attached to the 5′ ends of the two forward primers. One forward primer had a FAM fluorescent tag attached to its 5′ end, with the nucleotide sequence shown in SEQ ID NO.1: 5′-GAAGGTGACCAAGTTCATGCT-3′; the other forward primer had a HEX fluorescent tag attached to its 5′ end, with the nucleotide sequence shown in SEQ ID NO.2: 5′-GAAGGTCGGAGTCAACGGATT-3′. This resulted in a KASP molecular marker primer combination consisting of two fluorescently tagged forward competitive primers and one reverse universal primer, for use in... QTgw_2B High-throughput detection of excellent allelic variations at loci.

[0032] To further verify the accuracy and high resolution of the developed KASP molecular marker primer combination in recognizing target SNPs and detecting particle weight-enhancing sites, and to prove the successful development of KASP markers, it is necessary to validate the genotyping effect of KASP markers.

[0033] Specifically, the developed KASP molecular marker was used to analyze the Ningmai 9 × Yangmai 158 recombinant inbred line population that had passed chip detection. QTgw_2B The locus genotype was then used for reverse validation. If the KASP detection showed good specificity and amplification stability, clear clustering and typing, and the typing results were completely consistent with the microarray detection results, then the KASP marker was successfully developed and could be used for high-throughput detection of breeding materials and molecular marker-assisted breeding on a large scale. The specific method is as follows: 1) Preparation of DNA from test lines: Fifty materials were randomly selected from the recombinant self-pollination population of Ningmai 9 × Yangmai 158. Young leaves were taken from both parents and extracted using the CTAB method, referring to Stein et al. (2001). The DNA was then uniformly diluted to a concentration of about 120 ng / μL with sterile ultrapure water.

[0034] 2) Prepare the KASP reaction system: The total reaction system is 10 μL, including 2.5 μL of sample DNA at a concentration of 120 ng / μL, 5 μL of 2×KASP Master Mix, 0.14 μL of KASP Assay Mix, and 2.36 μL of ddH2O.

[0035] The KASP Assay Mix is ​​prepared as follows: Each 100 μL KASP Assay Mix contains 12 μL of each of two forward competitive primers at a concentration of 100 μM, 30 μL of a reverse universal primer at a concentration of 100 μM, and 46 μL of ddH2O.

[0036] 3) Set up the PCR reaction program: Step 1, heat activation at 94℃ for 15 min; Step 2, denaturation at 94℃ for 20 s, annealing and extension at 61-55℃ for 60 s (10 touch-down cycles, decreasing the temperature by 0.6℃ each cycle); Step 3, denaturation at 94℃ for 20 s, annealing and extension at 55℃ for 60 s, for 26 cycles. Finally, incubate the amplified products at 30℃.

[0037] 4) Fluorescence signal scanning and genotyping: The fluorescence signal of PCR amplification products was detected and genotyping was performed using an Applied Biosystems ABIViia7 Real Time PCR System (Thermo Scientific, USA). The genotyping results should meet the following requirements: parents with different genotypes should be clustered separately, the genotyping boundaries of recombinant inbred lines should be clear, the negative control should have no effective amplification signal, and the KASP genotyping results should be consistent with the chip detection results.

[0038] As described above, a set of sites that can be used for particle weight enhancement are obtained through development and design, manual evaluation, and genotyping detection. QTgw_2BThe KASP primer set for molecular detection (Table 1) is named KASP-2B_746101501 by the applicant. The KASP primer set specifically includes three primers: a forward competitive primer KASP-2B_746101501-1 with the nucleotide sequence shown in SEQ ID NO.3, a forward competitive primer KASP-2B_746101501-2 with the nucleotide sequence shown in SEQ ID NO.4, and a reverse universal primer KASP-2B_746101501-3 with the nucleotide sequence shown in SEQ ID NO.5.

[0039] The results show that ( Figure 2 (a) The KASP marker developed in this invention exhibits excellent genotyping performance. Specifically, when the wheat material clusters near the Y-axis and displays a blue FAM fluorescence signal in the genotyping clustering diagram, it is identified as the Yangmai 158 genotype; when it clusters near the X-axis and displays a red HEX fluorescence signal, it is identified as the Ningmai 9 genotype; samples clustered near the origin and displaying a black color serve as blank controls. The cluster boundaries between different genotypes are clear and the resolution is high. Furthermore, the detection results of this marker at the target SNP loci are completely consistent with the chip detection results of the corresponding parents and lines, indicating that the KASP marker has been successfully developed and can be used for wheat grain weight enhancement loci. QTgw_2B Genotyping and breeding selection.

[0040] Table 1 KASP primer design

[0041] Example 3: Application of the KASP molecular marker primer combination KASP-2B_746101501 To further verify the genotyping and application effects of KASP primer sets in wheat breeding materials, the applicant randomly selected 300 F7-F8 generation materials from high-generation wheat lines in the 2024-2025 breeding experiment of this institution as a verification population. The above materials were obtained by hybridization and selection of wheat varieties in the middle and lower reaches of the Yangtze River (including Huamai 2668, Huaimai 28, Wanmai 54, Xiangmai 25, Yangmai 11, Shengxuan 6, Anong 92484, Zhenmai 9, Huamai 5, Huamai 8, Zhen 261, Yangfumai 3, Sumai 188, Luomai 6010, Ruihua 306, Nannong 9918, etc.). The applicant self-numbered them as Line001-Line300, and the specific information is shown in Table 2. The above materials were planted at the Gaoyou Experimental Base in Jiangsu Province, and the thousand-grain weight was determined using the same method as in Example 1 for subsequent verification analysis.

[0042] Genomic DNA was extracted from the above materials and subjected to quantitative real-time PCR amplification and genotyping. The DNA extraction method, KASP reaction system, amplification procedure, and genotyping method were all consistent with those described in Example 2. The fluorescence detection results are as follows: Figure 2 As shown in Figure b. If the wheat material to be tested clusters close to the Y-axis in the genotyping clustering diagram and shows a blue FAM fluorescence signal, it is identified as the Yangmai 158 genotype and recorded as the AA genotype; if the wheat material to be tested clusters close to the X-axis and shows a red HEX fluorescence signal, it is identified as the Ningmai 9 genotype and recorded as the GG genotype. The specific genotype detection results for each material are shown in Table 2.

[0043] Table 2 Phenotypes and Genotypes of High Generation Wheat Breeding Lines

[0044] To verify the detection of KASP molecular markers in the breeding lines QTgw_2B The relationship between different genotypes at different loci and wheat grain weight phenotypes was investigated using SPSS 19.0 software for statistical analysis of the thousand-grain weight data of validation materials. The results showed that the average thousand-grain weight of the AA genotype materials was 47.00 g, while that of the GG genotype materials was 45.43 g; the AA genotype showed an increase of 1.57 g compared to the GG genotype, representing a growth rate of 3.46%. Figure 3 This indicates that materials carrying the AA genotype have higher grain weight performance.

[0045] The difference in thousand-grain weight between the two genotypes was further analyzed using an independent samples t-test. The results showed that the thousand-grain weight of the AA genotype was significantly higher than that of the GG genotype (t=3.748, p=2.14E-04) (Table 3).

[0046] Table 3 t-test results

[0047] Note: *** indicates a difference at a significance level of 0.001.

[0048] In summary, this invention identifies grain weight enhancement QTL loci based on genetic populations derived from the wheat backbone parents Ningmai 9 and Yangmai 158 in the middle and lower reaches of the Yangtze River wheat-growing region. QTgw_2B They also developed a set of KASP primers closely associated with this site. Carrying QTgw_2BThe AA genotype with enhanced allelic variation showed significantly increased grain weight, and related KASP markers enabled accurate genotyping of this superior allelic variation. These results were validated in both genetic populations and high-generation breeding lines, indicating that… QTgw_2B The loci and their KASP markers have good breeding applicability and can provide an effective tool for improving wheat grain weight and high-yield molecular breeding in the wheat-growing areas of the middle and lower reaches of the Yangtze River.

[0049] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0050] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0051] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. The application of a KASP molecular marker primer set in any of the following: (1) Detection of wheat grain weight enhancement sites QTgw_2B and the location of its related genes; (2) To detect or assist in the detection of wheat grain weight; (3) Breeding and creating high-grain-weight wheat varieties; (4) Sites that enhance wheat grain weight QTgw_2B Convergence breeding with other desirable trait sites; in, QTgw_2B Located on wheat chromosome 2B, the physical region on the Chinese spring reference genome is 716.55–746.10 Mb. The KASP molecular marker primer set includes primers with nucleotide sequences as shown in SEQ ID NO.3–SEQ ID NO.

5.

2. A method for detecting wheat grain weight, characterized in that, Includes the following steps: (1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using the KASP molecular marker primer set with nucleotide sequences as shown in SEQ ID NO.3-SEQ ID NO.5 to obtain the amplification product; (2) Fluorescence signal scanning and genotype clustering analysis were performed on the amplification products to determine the characteristics of the wheat material to be tested. QTgw_2B Genotype at the locus; (3) Determine the thousand-grain weight of the wheat material to be tested based on the genotype test results.

3. The method according to claim 2, characterized in that, In step (2), it is determined that the wheat material to be tested... QTgw_2B The method for determining the genotype at a locus is as follows: when the sample to be tested clusters near the Y-axis in the genotyping clustering diagram and displays a blue FAM fluorescence signal, the sample to be tested is determined to be of the AA genotype; when the sample to be tested clusters near the X-axis in the genotyping clustering diagram and displays a red HEX fluorescence signal, the sample to be tested is determined to be of the GG genotype.

4. The method according to claim 2, characterized in that, In step (3), the method for judging the thousand-grain weight of wheat is: the thousand-grain weight of wheat materials carrying the AA genotype is higher than that of wheat materials carrying the GG genotype.

5. A set of QTL loci associated with wheat grain weight QTgw_2B The relevant KASP molecular marker primer set is characterized by, QTgw_ 2B Located on wheat chromosome 2B, the physical region on the Chinese spring reference genome is 716.55–746.10 Mb. The KASP molecular marker primer set includes the forward competitive primer KASP-2B_746101501-1 with nucleotide sequence as shown in SEQ ID NO.3, the forward competitive primer KASP-2B_746101501-2 with nucleotide sequence as shown in SEQ ID NO.4, and the reverse universal primer KASP-2B_746101501-3 with nucleotide sequence as shown in SEQ ID NO.5.

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