KASP primer set related to wheat grain weight and its application

Abstract

The present application discloses a KASP primer set related to the wheat grain weight QTL site Qgw-7A and its application; Qgw-7A is located on wheat chromosome 7A, and its physical position on the Chinese Spring reference genome is 688.96 Mb-691.25 Mb; the QTL can significantly increase wheat grain weight; the KASP primer set consists of nucleotide sequences such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID The primers shown in NO.3 are KASP‑7A_688967553‑1, KASP‑7A_688967553‑2 and KASP‑7A_688967553‑3; the KASP primer set provides a molecular marker with simple and fast detection and accurate results for the screening of high-grain weight wheat. It can complete high-throughput screening of excellent genotype materials in the early generations or in the seedling stage when the grains have not yet formed. It is suitable for efficient detection of large-scale breeding lines, improves the efficiency of high-yield genetic improvement of wheat, and can also assist in the aggregation of excellent thousand-grain weight loci with other excellent traits, accelerating the selection and breeding of new wheat varieties with outstanding yield and excellent comprehensive traits.

Description

Technical Field

[0001] The invention relates to the field of wheat genetic breeding, and in particular to the development and application of a group of KASP markers related to a wheat grain weight QTL site Qgw-7A. Background Art

[0002] Wheat (Triticum aestivum L) is one of the most important food crops in the world and the staple food for 40% of the world's population (Pang Y, Liu C, Wang D, et al. (2020) High-resolution genome-wide association study identifies genomic regions and candidate genes for important agronomic traits in wheat. Mol Plant 13:1311-1327). As the world's population continues to increase, the global demand for wheat is also growing, and increasing food production has become an urgent task to achieve global food security.

[0003] The number of spikes per unit area, the number of grains per spike, and grain weight are three important factors that constitute wheat yield. However, the increase in the number of grains per spike is often accompanied by a decrease in the number of wheat spikes. In contrast, the increase in wheat grain weight is relatively independent. Grain weight is generally expressed as 1,000-grain weight, and its heritability is relatively large (Li T, Deng G, Su Y, et al. (2022) Genetic dissection of quantitative trait loci for grain size and weight by high-resolution genetic mapping in bread wheat (Triticum aestivum L.). Theor Appl Genet 135: 257–271). Therefore, while stabilizing the number of spikes and grains per spike, further increasing wheat grain weight is the main driving force for increasing wheat yield and is also the preferred factor for the selection of modern wheat fine varieties.

[0004] In recent years, the use of molecular biology to discover QTLs (Quantitative trait loci) and genes of target traits and develop molecular marker-assisted breeding methods that are closely linked to them has greatly replaced the traditional artificial breeding methods that are labor-intensive, slow, and inefficient, and has become an important means for breeders to accurately and efficiently select and target improve target traits at the genetic level (Würschum T. (2012) Mapping QTL for agronomic traits in breeding populations. Theor Appl Genet 125: 201-210). To date, many results have been achieved in the genetic research of wheat grain weight using forward and reverse genetics, through genetic map construction, genotyping, linkage or association analysis between genotypes and quantitative trait phenotypes. Many QTLs have been located, and some wheat grain weight-related genes and their variant types have been discovered and cloned one after another (Nadolska-Orczyk A, Rajchel I, Orczyk W, et al (2017) Major genes determining yield related traits in wheat and barley. Theor Appl Genet, 130: 1081-1098; Yang Y, Amo A, Wei D, et al. (2021) Large-scale integration of meta-QTL and genome-wide association study discovers the genomic regions and candidate genes for yield and yield-related traits in bread wheat. Theor Appl Genet 134: 3083-3109), which has greatly promoted the molecular breeding process of wheat grain weight. However, compared with other crops, the wheat genome is large and complex, and the genetic analysis of complex quantitative traits such as grain weight still faces difficulties, mainly manifested in the following ways: First, due to the influence of factors such as differences in parental genetic background, different QTL mapping methods, marker types and different natural environments, there are large differences in the QTL positioning results of wheat grain weight in different mapping populations; Second, the existing clear genetic loci, genes and molecular markers that can be used in breeding practice are far from meeting breeding needs.Therefore, constructing a high-quality genetic map, fully exploring the excellent grain weight-related QTLs of wheat genetic resources, especially the backbone wheat lines, and developing corresponding efficient and accurate molecular markers that can be used for breeding will be conducive to the screening, verification and utilization of excellent variation sites, thereby providing a beneficial driving force for molecular breeding of wheat yield.

[0005] KASP, or Kompetitive allele-specific PCR, is a new typing technology developed in recent years. This technology uses the specific matching of primer terminal bases to accurately type SNPs and InDels (Insertions or Deletions). Due to its batch, automated, and precise characteristics, it is particularly suitable for high-throughput detection of large-scale populations and is very consistent with breeding selection. It has become the mainstream molecular marker detection method in the world (Semagn K, Babu R, Hearne S, et al. (2014) Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement. Mol Breeding 33: 1-14). So far, KASP technology has been widely used in crop gene fine positioning, MAS (Molecular marker-assisted It has been widely used in fields such as molecular marker-assisted selection (Molecular marker-assisted selection) breeding (Yang Qingqing, Tang Jiaqi, Zhang Changquan, et al. (2022) Application and Prospect of KASP Marker Technology in Major Crops. Biotechnology Bulletin 38(04):58-71).

[0006] The wheat varieties Ningmai No. 9 and Yangmai 158 are high-yield, high-quality, disease-resistant wheat varieties bred by the Jiangsu Academy of Agricultural Sciences and the Lixiahe Regional Agricultural Science Research Institute of Jiangsu Province, respectively. They have outstanding characteristics in important traits such as grain weight and have dominated wheat production in the middle and lower reaches of the Yangtze River (for the definition of this wheat region, see Cheng Shunhe et al. (2012) (Cheng Shunhe, Guo Wenshan, Wang Longjun et al. (2012) "South China Wheat". Jiangsu Science and Technology Press). Varieties bred with them as the backbone parents, such as Yangmai 23 and Ningmai 14, are still the main varieties cultivated on a large scale in the middle and lower reaches of the Yangtze River. However, to date, there have been few reports on the genetic analysis of the grain weight of Ningmai 9 and Yangmai 158, as well as molecular markers suitable for high-throughput and accurate detection of excellent variations. Therefore, mining the QTL that controls grain weight and developing a new generation of high-throughput KASP molecular markers that can be used for breeding can not only provide new functional sites and gene resources for high-yield wheat breeding, but also can further combine molecular markers to efficiently and accurately identify excellent genotypes, providing a basis for the process of high-yield wheat breeding, including parent selection and offspring selection, so as to accelerate the breeding of breakthrough new high-grain weight wheat varieties, which is highly practical. Summary of the invention

[0007] In order to solve the problems existing in the above-mentioned technology, the present application designs a KASP primer set based on the QTL locus Qgw-7A that controls wheat grain weight, which is discovered for the first time, and applies the primers to the detection of wheat grain weight; the QTL locus Qgw-7A can significantly increase wheat grain weight, and the corresponding KASP marker can easily and accurately realize high-throughput identification and auxiliary selection of excellent genotype materials, which has a high application value for high-yield molecular breeding of wheat.

[0008] Specifically, the technical solutions provided by this application are as follows:

[0009] First, the present application provides a set of KASP primer sets related to wheat grain weight, which consists of a forward competitive primer KASP-7A_688967553-1 with a nucleotide sequence as shown in SEQ ID NO.1, a forward competitive primer KASP-7A_688967553-2 with a nucleotide sequence as shown in SEQ ID NO.2, and a reverse universal primer KASP-7A_688967553-3 with a nucleotide sequence as shown in SEQ ID NO.3.

[0010] The KASP primer set was developed based on the wheat grain weight QTL site Qgw-7A. Qgw-7A is located on chromosome 7A of wheat and its physical position on the Chinese Spring reference genome IWGSC RefSeq v1.0 is between 688.96Mb and 691.25Mb. The SNP molecular markers on both sides are 7A_688967553 and 7A_691259601, respectively.

[0011] The forward primers KASP-7A_688967553-1 and KASP-7A_688967553-2 of the KASP primer set have different fluorescent labels connected to their 5' ends. Among them, the 5' end of KASP-7A_688967553-1 is connected to a FAM fluorescent probe label (SEQ ID NO.7); the 5' end of KASP-7A_688967553-2 is connected to a HEX fluorescent probe label (SEQ ID NO.8)

[0012] Secondly, the present application also provides an application of the above-mentioned KASP primer set in detecting wheat grain weight, and the specific method comprises: using the genomic DNA of the wheat to be tested as a template, performing fluorescence quantitative PCR amplification using the above-mentioned KASP primers, and then using a fluorescence quantitative PCR instrument to perform fluorescence signal scanning on the amplified product, and performing genotyping on the wheat material to be tested according to the fluorescence signal: if the wheat material to be tested has a fluorescence probe signal of the forward primer KASP-7A_688967553-1 of the KASP primer set (a sample with a fluorescence signal value aggregated close to the Y axis and displayed in blue), but does not contain a fluorescence probe signal of the forward primer KASP-7A_688967553-2, it is considered that The wheat material to be tested contains the wheat grain weight QTL site Qgw-7A, which is recorded as genotype A (genotype type of Ningmai No. 9); if the wheat material to be tested has a fluorescent probe signal of the forward primer KASP-7A_688967553-2 of the KASP primer set (a sample with a fluorescent signal value aggregated close to the X-axis and displayed in red), but does not contain the fluorescent probe signal of the forward primer KASP-7A_688967553-1, it is considered that the wheat material to be tested does not contain the wheat grain weight QTL site Qgw-7A, which is recorded as genotype B (genotype type of Yangmai 158), and it is judged that the thousand-grain weight of the wheat strain of genotype A is significantly higher than that of the wheat strain of genotype B. The above-mentioned wheat variety to be tested is preferably wheat from the middle and lower reaches of the Yangtze River.

[0013] The total volume of the PCR amplification reaction system involved above is 10 μL, and its specific components include: 2.5 μL of genomic DNA with a concentration of 120 ng / ul, 5 μL of 2×KASP Master Mix (LGC), 0.14 μL of KASPAssay Mix, and 2.36 μL of ddH2O;

[0014] The preparation method of each 100 μL KASPAssay Mix is ​​as follows: 12 μL of the forward primer KASP-7A_688967553-1 with a concentration of 100 μM, 12 μL of the forward primer KASP-7A_688967553-2 with a concentration of 100 μM, 30 μL of the reverse universal primer KASP-7A_688967553-3 with a concentration of 100 μM, and 46 μL of ddH2O.

[0015] The PCR amplification reaction program was as follows: 1) 94°C heat activation for 15 min; 2) 94°C denaturation for 20 s, 61-55°C annealing extension for 1 min (10 touch-down cycles, 0.6°C lowered each cycle); 3) 94°C denaturation for 20 s, 55°C annealing extension for 1 min, 28 cycles. The amplified product was incubated at 30°C and the fluorescence signal was collected.

[0016] Third, the purpose of this application also includes the application of the provided KASP primer set, which includes any one of the following: 1) application in detecting the wheat grain weight QTL site Qgw-7A and the positioning of related genes; 2) application in breeding and creating high-grain weight wheat varieties; 3) application in promoting the aggregation breeding between the wheat grain weight QTL site Qgw-7A and other excellent trait sites.

[0017] The beneficial effects of this application are:

[0018] 1) In the present application, the backbone parents of wheat, Ningmai No. 9 and Yangmai 158, from the middle and lower reaches of the Yangtze River were used to construct a high-generation recombinant inbred population. A QTL locus Qgw-7A controlling wheat grain weight was finally located through phenotypic collection and linkage analysis. Qgw-7A is located on chromosome 7A, and its physical position is 688.96Mb-691.25Mb. This QTL can significantly increase wheat grain weight, which can not only provide new functional sites and gene resources for high-yield wheat breeding, but can also be further combined with molecular marker-assisted selection for transformation and utilization in breeding, thereby promoting the molecular improvement of grain weight traits, which has a high utilization value for high-yield wheat breeding.

[0019] 2) Furthermore, the present application has developed a KASP molecular marker and its application method for the QTL site Qgw-7A, which can be used to accurately and efficiently detect the grain weight QTLQgw-7A. The molecular marker is tightly linked to Qgw-7A, has the advantages of stable amplification, accurate typing, and easy and quick detection. It is very consistent with the demand for accurate detection of genotypes of large-scale genetic lines in wheat breeding, and can be used for high-throughput screening of wheat grain weight.

[0020] 3) The molecular marker primer combination provided in this application can promote the application of wheat grain weight QTL Qgw-7A in high-yield wheat breeding, realize the early identification and auxiliary selection of wheat with yield as the breeding target, improve the targeting and accuracy of breeding selection, and improve breeding efficiency; it can also assist in the aggregation of excellent thousand-grain weight loci with other excellent traits, and accelerate the breeding of new wheat varieties with outstanding yield and excellent comprehensive traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the genetic location of the QTL locus Qgw-7A controlling wheat grain weight.

[0022] Figure 2 This is a diagram showing the typing effect of the KASP molecular marker combination provided by the present invention and the genotype detection result of the verification material.

[0023] Among them, a is the typing effect diagram 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 material for trial verification; the coordinate axis value represents the allele fluorescence signal intensity, the black dot represents the negative control (NTC) (ie, ddH20), and the blue and red dots represent different genotypes, respectively.

[0024] Figure 3 This is a comparison of the grain weight of two different genotypes of the tested wheat materials detected by KASP molecular markers. Among them: A and B are two genotypes, and GW represents thousand-grain weight. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical content and advantages of the present invention more clear, the following embodiments are given in detail.

[0026] Unless otherwise specified, the terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art. In the embodiments, various processes and methods not described in detail are conventional methods known in the art.

[0027] Unless otherwise specified, the reagents, equipment, etc. used in the following examples can be obtained from commercial sources.

[0028] The wheat materials used in the present invention are all germplasm resources preserved in the provincial germplasm resource bank (crops) of Jiangsu Province - the College of Agriculture of Yangzhou University, and can be obtained and used by technicians and researchers in this field.

[0029] Example 1 Obtaining the wheat grain weight QTL locus Qgw-7A and marker interval

[0030] (1) In this example, wheat varieties Ningmai 9 were used as the female parent and Yangmai 158 as the male parent to obtain the F1 generation, which was then self-pollinated to obtain the F2 generation. A high-generation recombinant inbred line genetic population containing 282 strains was obtained through the single-seed descent method.

[0031] (2) The whole genome of a recombinant inbred population consisting of 282 lines constructed from Ningmai 9×Yangmai 158 was scanned using the Illumina 90k gene chip to construct a linkage genetic map. The genetic linkage map covers 21 chromosomes of wheat and has a total length of 3022 cM.

[0032] (3) The above-mentioned recombinant self-pollinated population was planted at the experimental base of Yangzhou University (119.40°E, 32.34°N) for two consecutive years in 2020-2021 and 2021-2022. Single-row sowing was carried out with a row length of 1.2 m and a row spacing of 0.3 m, and three replicates. The soil conditions in the experimental base were uniform. Before harvesting, the field management was completely consistent with the method published by Ma Hongxiang et al. (2021) (Ma Hongxiang, Gu Kejun, Chen Huaigu (2021) "100 Questions on Key Practical Technologies in Wheat Industry", China Agricultural Press.). When the grains were mature, 5 plants with uniform growth were randomly harvested from each line. After the grains were threshed and naturally dried, 1,000 seeds were randomly selected from them to measure their weight. The thousand-grain weight represented the grain weight, and the average value was used for data analysis.

[0033] In addition, 213 wheat samples were randomly selected from the high-generation (F7-F8) wheat breeding line materials obtained by the applicant using wheat varieties in the middle and lower reaches of the Yangtze River (including Zhengmai 9023, Huamai 2668, Huaimai 28, Wanmai 54, Xiangmai 25, Yangmai 11, Shengxuan 6, Annong 92484, Zhenmai 9, Huamai 5, Huamai 8, Zhen 261, Yangfumai 3, Sumai 188, Luomai 6010, Ruihua 306, Nannong 9918, etc.) (the applicant named the material Line001-Line213, as shown in Table 2), and their thousand-grain weight was measured by the same method for subsequent verification.

[0034] (4) Combined genotype and phenotype data, QTL IciMapping v4 software was used to locate the QTL for grain weight (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), and the complete interval mapping method (ICIM-ADD) was used, with a LOD value of 2.5 as the threshold of significance. Finally, a stable QTL that increased grain weight from Ningmai No. 9 and was detected in different environments was located. The applicant named it Qgw-7A, and its genetic location is as follows Figure 1 Qgw-7A is located on wheat chromosome 7A, and the SNP molecular markers on both sides are 7A_688967553 and 7A_691259601, respectively. The physical position of Qgw-7A on the Chinese Spring reference genome IWGSC RefSeq v1.0 is between 688.96Mb and 691.25Mb.

[0035] Example 2 Development, screening and validation of KASP molecular markers

[0036] (1) In order to better utilize the grain weight locus Qgw-7A screened in Example 1 for breeding, KASP markers suitable for high-throughput typing were further developed based on its interval marker SNP composition. As shown in Table 1, the SNPs are 7A_688967553 and 7A_691259601. First, the sequence information of 150 bp upstream and downstream of each SNP was retrieved respectively, and then Primer Premier5 (http: / / www.premierbiosoft.com / primerdesign / ) was used to design multiple sets of amplification primers. Then, DNAMAN was used to evaluate the quality of the designed primers. The Ensembl Plants database was further used to detect primer specificity. Finally, the screened primer sequences were converted into KASP primers (Yang Qingqing, Tang Jiaqi, Zhang Changquan, et al. (2022) Application and Prospect of KASP Marker Technology in Major Crops. Biotechnology Bulletin 38(04):58-71). The KASP primer sequences involved are shown in Table 1. Each pair of primers consists of three sequences, namely: forward competitive primer 1: FAM tag sequence + amplification primer sequence; forward competitive primer 2: HEX tag sequence + amplification primer sequence; reverse universal primer: amplification primer sequence; among which, the nucleotide sequences of FAM tag sequence and HEX tag sequence are as follows:

[0037] FAM tag sequence (SEQ ID NO.7): 5'-GAAGGTGACCAAGTTCATGCT-3' (can bind to FAM fluorescent group);

[0038] HEX tag sequence (SEQ ID NO. 8): 5'-GAAGGTCGGAGTCAACGGATT-3' (can bind to HEX fluorescent group).

[0039] Table 1 KASP primer design

[0040]

[0041] Note: In the SNP composition (a / b), a represents the variation type of Ningmai 9, and b represents the variation type of Yangmai 158.

[0042] (2) Fifty accessions were randomly selected from the Ningmai 9×Yangmai 158 recombinant self-pollinated population. Young leaves were taken from the two parents. Genomic DNA was extracted from the accessions using the CTAB method according to Stein et al. (2001) (Stein N, Herren G, and Keller B (2001) A new DNA extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum. Plant Breed 120:354-356). The DNA was then diluted to a uniform concentration of about 120 ng / ul using sterile ultrapure water.

[0043] Fluorescence quantitative PCR amplification was performed using the KASP primer set obtained in Table 1. The reaction system, amplification procedure and genotyping method were as follows:

[0044] 1) KASP reaction system: The total volume was 10 μL, including 2.5 μL of sample DNA (120 ng / ul), 5 μL of 2×KASP Master Mix (LGC Genomics, Hoddeston, UK), 0.14 μL of KASP Assay Mix, and 2.36 μL of ddH2O.

[0045] The preparation method of KASPAssay Mix is ​​as follows: each 100 μL KASPAssay Mix contains 12 μL of each of two forward competitive primers with a concentration of 100 μM, 30 μL of a reverse universal primer with a concentration of 100 μM, and 46 μL of ddH2O.

[0046] 2) KASP reaction procedure: 94℃ heat activation for 15min; 94℃ denaturation for 20s, 61~55℃ annealing and extension for 1min (10 touch-down cycles, 0.6℃ lowered each cycle); 94℃ denaturation for 20s, 55℃ annealing and extension for 1min, 28 cycles. Amplification products were kept at 30℃ and fluorescence signals were collected.

[0047] 3) Genotyping: The fluorescence signal was scanned and genotyped using a fluorescence quantitative PCR instrument Applied Biosystems ABI Viia7 Real Time PCR System (Thermo Scientific, USA). Specifically, the fluorescence type of the sample whose fluorescence signal intensity value is aggregated close to the Y-axis and displays blue is the FAM fluorescent group type, which is the Ningmai No. 9 genotype type; the fluorescence type of the sample whose fluorescence signal intensity value is aggregated close to the X-axis and displays red is the HEX fluorescent group type, which is the Yangmai 158 genotype type; the sample that is aggregated close to the origin and displays black is the blank control.

[0048] According to the typing results, a group of KASP primers with high amplification efficiency, clear typing, and complete consistency with the corresponding genotype parent clustering were finally screened out (such as Figure 2 The applicant named the primer set KASP-7A_688967553. The nucleotide sequence of the KASP primer set includes

[0049] The three sequences, KASP-7A_688967553-1 (SEQ ID NO.1), KASP-7A_688967553-2 (SEQ ID NO.2) and KASP-7A_688967553-3 (SEQ ID NO.3), can be applied to the detection and breeding selection of wheat grain weight locus Qgw-7A, wherein the dominant allele variation is derived from Ningmai No. 9, and the non-dominant allele variation is derived from Yangmai 158.

[0050] Example 3 Application of KASP-7A_688967553 Molecular Marker

[0051] Using the KASP primer combination KASP-7A_688967553 obtained in Example 2, genomic DNA was extracted from 213 randomly selected wheat advanced breeding line materials (i.e., Line001-Line213 in Table 2) and two parents, and fluorescence quantitative PCR amplification and genotyping were performed. DNA extraction, KASP reaction system, amplification procedure and genotyping method were consistent with the method described in Example 2. The fluorescence detection results are shown in FIG. Figure 2 As shown in b.

[0052] The final results showed that the fluorescent signal with the same fluorescence signal as Ningmai 9 was recorded as genotype A, and the fluorescent signal with the same fluorescence signal as Yangmai 158 was recorded as genotype B (Table 2).

[0053] Table 2 Phenotypes and genotypes of advanced wheat breeding lines

[0054]

[0055]

[0056]

[0057] SPSS 19.0 was used for statistical analysis. Comparing the grain weight phenotype data of genotypes A and B in Table 2, and the grain weight of materials carrying these two genotypes, it was found that the grain weight of wheat carrying genotype A was higher than that of wheat carrying genotype B ( Figure 3 ).

[0058] The t-test further showed that the phenotypic difference between the high-grain weight genotype (genotype A) and the low-grain weight genotype (genotype B) reached an extremely significant level (Table 3).

[0059] Table 3 t-test results

[0060]

[0061]

[0062] Note: * indicates a difference at a significance level of 0.05, and ** indicates a difference at a significance level of 0.01

[0063] It can be seen that the genotype (A genotype) carrying the provided QTL locus Qgw-7A can significantly increase the weight of wheat grains, and the KASP marker related thereto can complete the accurate typing of the dominant allele variation. The test wheat in the above embodiment includes population materials derived from the backbone wheat varieties Yangmai 158 and Ningmai No. 9 in the middle and lower reaches of the Yangtze River as parents, and wheat high-generation (F7-F8) breeding lines obtained by hybridizing varieties in the middle and lower reaches of the Yangtze River wheat area. Therefore, the identified QTL locus Qgw-7A and the developed KASP molecular markers have high application value for wheat, especially for high-yield molecular breeding of wheat in the middle and lower reaches of the Yangtze River.

Claims

1. A KASP primer set related to wheat grain weight, It is characterized in that The KASP primer set consists of a forward primer KASP-7A_688967553-1 having a nucleotide sequence as shown in SEQ ID NO.1, a forward primer KASP-7A_688967553-2 having a nucleotide sequence as shown in SEQ ID NO.2, and a reverse universal primer KASP-7A_688967553-3 having a nucleotide sequence as shown in SEQ ID NO.

3.

2. Use of the KASP primer set as claimed in claim 1 in detecting wheat grain weight.

3. The use according to claim 2, It is characterized in that The specific steps are as follows: using the genomic DNA of the wheat to be tested as a template, using the KASP primer set to perform fluorescence quantitative PCR amplification to obtain an amplified product; then performing fluorescence signal scanning and genotyping on the amplified product, if the wheat to be tested has a fluorescence signal of the forward primer KASP-7A_688967553-1, but does not have a fluorescence signal of the forward primer KASP-7A_688967553-2, the wheat is recorded as genotype A; if the wheat to be tested has a fluorescence signal of the forward primer KASP-7A_688967553-2, but does not have a fluorescence signal of the forward primer KASP-7A_688967553-1, the wheat material is recorded as genotype B; the grain weight of genotype A wheat is higher than that of genotype B wheat.

4. The use according to claim 3, It is characterized in that The fluorescent quantitative PCR amplification refers to: PCR amplification system: 2.5 μL of template DNA with a concentration of 120 ng / ul, 5 μL of 2×KASP Master Mix, 0.14 μL of KASPAssay Mix, ddH 2 O was added to make up to 10 μL; The preparation method of each 100 μL of KASP Assay Mix is ​​as follows: 12 μL of the forward primer KASP-7A_688967553-1 with a concentration of 100 μM, 12 μL of the forward primer KASP-7A_688967553-2 with a concentration of 100 μM, 30 μL of the reverse universal primer KASP-7A_688967553-3 with a concentration of 100 μM, ddHO 2 O 46 μL; PCR amplification program: 94℃ heat activation for 15 min; 94℃ denaturation for 20 s, 61-55℃ annealing and extension for 1 min, including 10 touch-down cycles, with each cycle decreasing by 0.6℃; 94℃ denaturation for 20 s, 55℃ annealing and extension for 1 min, 28 cycles; finally, keep at 30℃ and collect fluorescence signals.

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