Specific primers of molecular markers linked to genes for traits associated with wheat kernels and use thereof

Abstract

The present application relates to the field of genetic breeding, in particular to specific primers of molecular markers linked to wheat grain-related traits and application. The present application obtains molecular markers closely linked to grain-related trait genes, which are obtained by whole genome association analysis on grain-related traits of Sichuan germplasm population composed of local varieties and bred varieties in Sichuan region of China. The markers are close to or directly located on the related genes of grain-related traits, have good repeatability and strong stability, and can be used for identification of wheat grain-related traits and molecular assisted selection of wheat offspring with excellent grain traits, thereby accelerating the breeding process of excellent grain wheat varieties.

Description

Technical Field

[0001] The present invention relates to the field of genetic breeding, and in particular to specific primers of molecular markers linked to wheat grain-related trait genes and applications thereof. Background Art

[0002] Wheat grain-related traits include average grain length, average grain width, average grain area, average grain circumference, average grain length-to-width ratio and 1000-grain weight. Materials with excellent grain-related traits are of great significance to wheat yield breeding.

[0003] Recent advances in molecular biology and sequencing technologies have led to progress in homologous cloning and molecular marker development for wheat grain weight-related genes. Using homologous cloning, an increasing number of genes associated with 1000-grain weight have been cloned, providing valuable insights for marker-assisted selection breeding. Due to the complexity of the wheat genome, only a subset of genes associated with grain-related traits have been identified through homologous cloning.

[0004] Using genome-wide association analysis to precisely locate genes for grain-related traits allows for the screening and effective utilization of genetic material with superior grain-related traits. Gene mapping using molecular markers offers the advantages of simplicity, rapidity, and precision. Single nucleotide polymorphism (SNP) markers are much more diverse than earlier molecular markers based on DNA hybridization and polymerase chain reaction. Microarray-based genotyping arrays with a large number of single nucleotide polymorphism probes are now available as Illumina Infinium BeadChip or Affymetrix Axiom arrays, allowing for robust and cost-effective genotyping of breeding material. The integration of SNP marker technology with DNA microarray and chip technologies has made SNP marker technology the most promising third-generation molecular marker after RFLP and SSR markers. It plays an important role in crop breeding, including genetic map construction, germplasm resource analysis, biodiversity detection, and linkage disequilibrium association analysis. Summary of the Invention

[0005] The purpose of the present invention is to provide specific primers for molecular markers linked to genes related to wheat excellent grain traits.

[0006] Another object of the present invention is to provide the use of specific primers for the above-mentioned molecular markers linked to genes related to wheat excellent grain traits.

[0007] Another object of the present invention is to provide a method for identifying wheat varieties with excellent grain-related traits.

[0008] According to the technical solution of the present application, molecular markers linked to genes related to wheat excellent grain traits were obtained by typing using KASP primers KASP-AX-108776765, KASP-AX-110419159, KASP-AX-110385388, and KASP-AX-110488530. They were located on wheat chromosomes 2B, 2D, and 6B, respectively, with a physical distance of 1.92 Mb.

[0009] The KASP primer KASP-AX-108776765 includes:

[0010] Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTTGATTTCTAGAATAAAACGTTCCCATTCTG-3' (SEQ ID NO: 1), forward primer B: 5'-GAAGGTCGGAGTCAACGGATTTGATTTCTAGAATAAAACGTTCCCATTCC-3' (SEQ ID NO: 2), reverse primer: 5'-GGCCCCTTACAAATACAGTATAAAACA-3' (SEQ ID NO: 3);

[0011] The KASP primer KASP-AX-110419159 includes:

[0012] Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTGCCAAACCGTGAGCCTAATAA-3' (SEQ ID NO: 4), forward primer B: 5'-GAAGGTGACCAAGTTCATGCTGCCAAACCGTGAGCCTAATAT-3' (SEQ ID NO: 5), reverse primer: 5'-AGTGCTGCTGTAATTCTTTCCC-3' (SEQ ID NO: 6);

[0013] The KASP primer KASP-AX-110385388 includes:

[0014] Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTCCGGTGAGGGTTTTAATCACATG-3' (SEQ ID NO: 7), forward primer B: 5'-GAAGGTCGGAGTCAACGGATTCCGGTGAGGGTTTTAATCACATC-3' (SEQ ID NO: 8), reverse primer: 5'-CCTTGCTGGCTATGACTACCC-3' (SEQ ID NO: 9);

[0015] The KASP primer KASP-AX-110488530 comprises:

[0016] Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTTCACCACTGGAGAAAACCGT-3' (SEQ ID NO: 10), forward primer B: 5'-GAAGGTCGGAGTCAACGGATTTCACCACTGGAGAAAACCGC-3' (SEQ ID NO: 11), reverse primer: 5'-CACCAGTGATGGCTTTACGTAAT-3' (SEQ ID NO: 12).

[0017] The method for identifying a gene related to excellent grain traits in wheat according to the present application comprises the step of amplifying the material to be detected using KASP primers, wherein the KASP primers comprise:

[0018] Primer KASP-AX-108776765:

[0019] Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTTGATTTCTAGAATAAAACGTTCCCATTCTG-3',

[0020] Forward primer B: 5'-GAAGGTCGGAGTCAACGGATTTGATTTCTAGAATAAAACGTTCCCATTCC-3',

[0021] Reverse primer: 5'-GGCCCCTTACAAATACAGTATAAAACA-3';

[0022] Primer KASP-AX-110419159:

[0023] Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTGCCAAACCGTGAGCCTAATAA-3',

[0024] Forward primer B: 5'-GAAGGTGACCAAGTTCATGCTGCCAAACCGTGAGCCTAATAT-3',

[0025] Reverse primer: 5'-AGTGCTGCTGTAATTCTTTCCC-3';

[0026] Primer KASP-AX-110385388:

[0027] Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTCCGGTGAGGGTTTTAATCACATG-3',

[0028] Forward primer B: 5'-GAAGGTCGGAGTCAACGGATTCCGGTGAGGGTTTTAATCACATC-3',

[0029] Reverse primer: 5′-CCTTGCTGGCTATGACTACCC-3′;

[0030] Primer KASP-AX-110488530:

[0031] Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTTCACCACTGGAGAAAACCGT-3',

[0032] Forward primer B: 5'-GAAGGTCGGAGTCAACGGATTTCACCACTGGAGAAAACCGC-3', reverse primer: 5'-CACCAGTGATGGCTTTACGTAAT-3'.

[0033] According to the method for identifying genes related to excellent wheat grain traits of the present invention, the FAM signal marker is connected to the forward primer A, and the HEX signal marker is connected to the forward primer B.

[0034] According to the method for identifying genes related to excellent wheat grain traits of the present invention, the blue fluorescence type 1 in KASP-AX-108776765 carries grain-related trait genes and can significantly promote grain-related traits such as wheat grain length, width and 1000-grain weight; the orange-yellow fluorescence is type 2, which does not carry grain-related trait genes.

[0035] The blue fluorescence type 1 in KASP-AX-110419159 carries grain-related trait genes and can significantly promote grain-related traits such as wheat grain length, width and 1000-grain weight; the orange-yellow fluorescence is type 2, which does not carry grain-related trait genes.

[0036] The orange-yellow fluorescence type 1 in KASP-AX-110385388 carries grain-related trait genes and can significantly promote grain-related traits such as wheat grain length, width and 1000-grain weight; the blue fluorescence is type 2, which does not carry grain-related trait genes.

[0037] KASP-AX-110488530 is orange yellow fluorescence type 1, carries grain-related trait genes, and can significantly promote grain-related traits such as length, width and thousand-grain weight of wheat grain; blue fluorescence is type 2, and does not carry grain-related trait genes.

[0038] The positive effects and applications of the present application are as follows:

[0039] The molecular markers obtained by the present application and closely linked to the excellent grain-related trait genes, specifically related to the traits of average grain length, average grain width, average grain area, average grain circumference, average grain length-width ratio and thousand-grain weight, are a plurality of new markers obtained by performing whole genome association analysis on the grain-related traits of a Sichuan germplasm population composed of local varieties and bred varieties in Sichuan, China. These markers are close to or directly located on the related genes of the grain-related traits, have good repeatability and strong stability, and can be used for identification of wheat grain-related traits and molecular-assisted selection of wheat excellent grain trait offspring, thereby accelerating the breeding process of wheat varieties with excellent grain-related traits. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 KASP-AX-108776765 in the Sichuan wheat germplasm population is shown, wherein orange yellow is type 1, blue is type 2, and black is a blank control;

[0041] Figure 2 KASP-AX-108776765 in the Sichuan wheat germplasm population BLUE value is shown, wherein the difference between the two types in different grain-related traits is shown;

[0042] Figure 3 KASP-AX-110419159 in the Sichuan wheat germplasm population is shown, wherein orange yellow is type 1, blue is type 2, and black is a blank control;

[0043] Figure 4 KASP-AX-110419159 in the Sichuan wheat germplasm population BLUE value is shown, wherein the difference between the two types in different grain-related traits is shown;

[0044] Figure 5 KASP-AX-110385388 in the Sichuan wheat germplasm population is shown, wherein orange yellow is type 1, blue is type 2, and black is a blank control;

[0045] Figure 6 KASP-AX-110385388 in the Sichuan wheat germplasm population BLUE value is shown, wherein the difference between the two types in different grain-related traits is shown;

[0046] Figure 7 Shows the typing results of KASP-AX-110488530 in the Sichuan wheat germplasm population, where orange represents type 1, blue represents type 2, and black represents the blank control;

[0047] Figure 8 It shows the differences between the two types in grain-related traits of KASP-AX-110488530 in the BLUE value of Sichuan wheat germplasm population;

[0048] Figure 9 The differences in grain-related traits among the BLUE values ​​of five different marker combinations in Sichuan wheat germplasm populations were shown. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below with reference to the embodiments, but is not intended to limit the present invention. Any equivalent substitutions in the art made according to the disclosure of the present invention shall fall within the scope of protection of the present invention.

[0050] Example 1 SNP molecular markers linked to genes related to wheat quality grain traits

[0051] Acquisition and Validation of KASP-AX-108776765, KASP-AX-110419159, and KASP-AX-110488530

[0052] 1. Test materials

[0053] Plant material:

[0054] A total of 233 Sichuan wheat varieties were collected, including 77 local varieties and 156 developed varieties. The 77 local germplasms were primarily provided by the China National Germplasm Bank and the Wheat Research Institute of Sichuan Agricultural University (with the germplasm code AS). The 156 developed germplasms were approved by Sichuan Province between 1997 and 2016 and were primarily developed and provided by research institutes such as the Sichuan Academy of Agricultural Sciences, the Mianyang Academy of Agricultural Sciences, the Chengdu Institute of Biology, Chinese Academy of Sciences, the Xichang Institute of Agricultural Sciences, Sichuan Agricultural University, and Southwest University of Science and Technology.

[0055] 2. Phenotypic identification

[0056] Field Experiment Design: Sichuan local wheat populations were planted in November 2018, November 2019, and November 2020 at the Wenjiang Teaching Experimental Base (E1, E4, E7), Chongzhou Modern Agriculture Teaching Base (E2, E5, E8), and Ya'an Experimental Teaching Base (E3, E6, E9) of Sichuan Agricultural University, respectively. Furthermore, the experiment was planted in November 2020 at the Chengdu Plain Agricultural Ecological Experimental Station of the Chinese Academy of Sciences in Shifang (E10). Field rows were 2 m long, with 0.3 m spacing between rows and 0.1 m between plants. Each material was planted in a single row, with single seeds sown in a single nest. Field management was the same as for field production.

[0057] After harvest, grain traits were measured in accordance with the common wheat trait testing standard GB / T 19557.2-2004 for the test materials grown under different field conditions. The average values ​​were then used to represent the phenotype of the line. At least 100 harvested, dried seeds were placed on a GreenBio seed scanner (model LBKZ-II) to obtain grain trait data. Data were then processed using Microsoft Office Excel 2019 to obtain phenotypic data for six grain-related traits: average kernel length, average kernel width, kernel width-to-length ratio, average kernel circumference, average kernel area, and 1000-kernel weight. The best linear unbiased estimator (BLUE) was then calculated.

[0058] 3. Phenotype-genotype association analysis

[0059] The mixed linear model (Q+K) in Tassel v5.2.38 software was used to analyze the association between grain traits and genotypes in 233 Sichuan wheat accessions. After quality control, effective SNP molecular markers were obtained for genome-wide association analysis.

[0060] 4. Genome-wide significant association site analysis

[0061] In the genome-wide association analysis, markers with -log10(P) ≥ 4 were considered significantly associated. Adjacent significantly associated loci were determined to belong to the same QTL block based on whether they were within the linkage disequilibrium (LD) distance.

[0062] In subsequent analysis, the physical positions of markers were determined based on the genes and QTLs reported to be related to agronomic traits and grain traits, using the Chinese Spring genome reference map IWGSC RefSeq v1.0 as a reference. Molecular markers within the linkage disequilibrium decay distance range were defined as the same QTL interval and compared with the reported grain trait sites to determine whether they were new QTLs.

[0063] 5. Tag development

[0064] Based on the preliminary GWAS positioning results, the located SNP marker sites were converted into KASP markers to track the main effect QTL. A total of 4 markers: KASP-AX-108776765, KASP-AX-110419159, KASP-AX-110385388 and KASP-AX-110488530, were located on chromosomes 2B, 2D and 6B, respectively. According to the linkage disequilibrium decay distance, the physical distance of each marker was 1.92 Mb.

[0065] KASP primers were designed and optimized to include:

[0066] KASP-AX-108776765:

[0067] Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTTGATTTCTAGAATAAAACGTTCCCATTCTG-3',

[0068] Forward primer B: 5'-GAAGGTCGGAGTCAACGGATTTGATTTCTAGAATAAAACGTTCCCATTCC-3', reverse primer: 5'-GGCCCCTTACAAATACAGTATAAAACA-3'.

[0069] KASP-AX-110419159:

[0070] Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTGCCAAACCGTGAGCCTAATAA-3',

[0071] Forward primer B: 5'-GAAGGTGACCAAGTTCATGCTGCCAAACCGTGAGCCTAATAT-3',

[0072] Reverse primer: 5'-AGTGCTGCTGTAATTCTTTCCC-3'.

[0073] KASP-AX-110385388:

[0074] Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTCCGGTGAGGGTTTTAATCACATG-3',

[0075] Forward primer B: 5'-GAAGGTCGGAGTCAACGGATTCCGGTGAGGGTTTTAATCACATC-3', reverse primer: 5'-CCTTGCTGGCTATGACTACCC-3'

[0076] KASP-AX-110488530:

[0077] Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTTCACCACTGGAGAAAACCGT-3',

[0078] Forward primer B: 5'-GAAGGTCGGAGTCAACGGATTTCACCACTGGAGAAAACCGC-3',

[0079] Reverse primer: 5'-CACCAGTGATGGCTTTACGTAAT-3'.

[0080] FAM and HEX signals were added to the primers to distinguish the two genotypes. The KASP assay results were detected using the Bio-Rad CFX96 real-time PCR system to obtain genotype data.

[0081] 6. Typing of molecular markers KASP-AX-108776765, KASP-AX-110419159, KASP-AX-110385388, and KASP-AX-110488530 in Sichuan germplasm populations

[0082] KASP-AX-108776765: Based on the typing results, the population can be divided into two types, Type 1 and Type 2. Among them, 60 wheat varieties showed blue fluorescence in the test, which is Type 1, 172 materials were identified as orange-yellow fluorescence, which is Type 2, and 1 material could not be classified (the result is as follows Figure 1 ). T test of grain related traits was conducted on the BLUE values ​​of type 1 and type 2 (the results are shown in Figure 2 ), and analyzed its significance. It can be found that in the BLUE value, the materials carrying genes for excellent grain-related traits, namely type 1, have extremely significantly higher (P=0<0.01) grain-related traits except grain length-to-width ratio than the materials not carrying genes for grain-related traits, namely type 2.

[0083] KASP-AX-110419159: Based on the typing results, the population can be divided into two types, Type 1 and Type 2. Among them, 170 wheat varieties showed blue fluorescence in the test, which is Type 1, 55 materials were identified as orange-yellow fluorescence, which is Type 2, and 12 materials could not be classified (the results are as follows Figure 3 ). T test of grain related traits was conducted on the BLUE values ​​of type 1 and type 2 (the results are shown in Figure 4), and analyzed its significance. It was found that in the BLUE value, the materials carrying genes for excellent grain-related traits, namely type 1, had significantly higher (P=0<0.01) grain-related traits except 1000-grain weight than the materials not carrying genes for grain-related traits, namely type 2. In terms of 1000-grain weight, type 1 was significantly higher (P=0<0.05) than type 2.

[0084] KASP-AX-110385388: Based on the typing results, the population can be divided into two types, Type 1 and Type 2. Among them, 166 wheat varieties showed orange-yellow fluorescence in the test, which is Type 1, and 67 materials were identified as blue fluorescence, which is Type 2 (the results are as follows Figure 5 ). T test of grain related traits was conducted on the BLUE values ​​of type 1 and type 2 (the results are shown in Figure 6 ), and analyzed its significance. It can be found that in the BLUE value, the materials carrying genes for excellent grain-related traits, namely type 1, have significantly higher (P=0<0.05) higher grain-related traits than the materials not carrying genes for grain-related traits, except for the average grain width and the average grain length-to-width ratio, while the other four grain-related traits are extremely significantly (P=0<0.01) higher than those of the materials not carrying genes for grain-related traits, namely type 2.

[0085] KASP-AX-110488530: Based on the typing results, the population can be divided into two types, Type 1 and Type 2. Among them, 83 wheat varieties showed orange-yellow fluorescence during the test, which is Type 1, 148 materials were identified as blue fluorescence, which is Type 2, and 2 materials could not be classified (the results are as follows Figure 9 ). T test of grain related traits was conducted on the BLUE values ​​of type 1 and type 2 (the results are shown in Figure 8 ), and analyzed its significance. It can be found that in the BLUE value, the materials carrying genes for excellent grain-related traits, namely type 1, have extremely significantly higher (P=0<0.01) grain-related traits than the materials not carrying genes for grain-related traits, namely type 2.

[0086] In summary, all four KASP markers can be used to screen for genes associated with superior grain traits in wheat varieties, providing a tool for subsequent molecular breeding of wheat grain-related traits.

[0087] 7. Comparison of molecular marker combinations of KASP-AX-108776765, KASP-AX-110419159, KASP-AX-110385388, and KASP-AX-110488530 in different types of Sichuan germplasm populations

[0088] A total of 8 combination types were obtained by combining the 4 KASP markers obtained. The combination types with more than 10 wheat varieties in a single combination type were further analyzed and counted. A total of 5 combination types contained more than 10 wheat varieties, namely type 1222 (KASP-AX-108776765 type 1; KASP-AX-110419159 type 2; KASP-AX-110385388 type 2; KASP-AX-110488530 type 2), type 2111 (KASP-AX-108776765 type 2; KASP-AX-110419159 type 1; KASP-AX-110385388 type 1; KASP-AX-110488530 type 1), and type 2112 (KASP-AX-108776765 type 2; KASP-AX-110419159 type 1; KASP-AX-110385388 type 1; KASP-AX-110488530 type 2), type 2121 (KASP-AX-108776765 type 2; KASP-AX-110419159 type 1; KASP-AX-110385388 type 2; KASP-AX-110488530 type 1), and type 2122 (KASP-AX-108776765 type 2; KASP-AX-110419159 type 1; KASP-AX-110385388 type 2; KASP-AX-110488530 type 2). The five combination types were tested for differences by data analysis (the results are shown in the figure). Figure 9 ) and are identified by different letters. Different lowercase letters indicate significant differences, while identical lowercase letters indicate insignificant differences. Comparison revealed that type 1222 performed significantly worse than types 2111, 2112, and 2121 in all six grain-related traits, with type 2111 performing best. Therefore, multiple marker combination types can more accurately screen wheat varieties for grain-related traits. Combination detection of four molecular markers can be used to predict the presence of genes for superior grain-related traits in wheat materials and applied to molecular breeding for wheat grain-related traits.

[0089] The above embodiments are only used to explain the technical solutions of the present application and do not limit the scope of protection of the present application.

Claims

1. A specific primer set for molecular markers linked to wheat grain-related trait genes, characterized in that: The specific primer set includes the following KASP primer pair: KASP-AX-108776765 Primer pairs ， The following primer pairs are included: Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTTGATTTCTAGAATAAAACGTTCCCATTCTG-3', forward primer B: 5'-GAAGGTCGGAGTCAACGGATTTGATTTCTAGAATAAAACGTTCCCATTCC-3', Reverse primer: 5′-GGCCCCTTACAAATACAGTATAAAACA-3′; KASP-AX-110419159 Primer pairs ， The following primers are included: Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTGCCAAACCGTGAGCCTAATAA-3', Forward primer B: 5'-GAAGGTCGGAGTCAACGGATTGCCAAACCGTGAGCCTAATAT-3', Reverse primer: 5′-AGTGCTGCTGTAATTCTTTCCC-3′; KASP-AX-110385388 Primer pairs ， The following primers are included: Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTCCGGTGAGGGTTTTAATCACATG-3', Forward primer B: 5'-GAAGGTCGGAGTCAACGGATTCCGGTGAGGGTTTTAATCACATC-3', Reverse primer: 5'-CCTTGCTGGCTATGACTACCC-3'; KASP-AX-110488530 Primer pairs ， The following primers are included: Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTTCACCACTGGAGAAAACCGT-3', Forward primer B: 5'-GAAGGTCGGAGTCAACGGATTTCACCACTGGAGAAAACCGC-3', Reverse primer: 5'-CACCAGTGATGGCTTTACGTAAT-3'.

2. Use of the specific primer set for the molecular marker linked to the wheat grain-related trait gene according to claim 1 in identifying wheat grain-related traits, wherein: Wheat grain-related traits include average grain length, average grain width, grain width-to-length ratio, average grain circumference, average grain area and 1000-grain weight.

3. A method for identifying wheat grain-related trait genes, characterized in that: The method comprises the step of amplifying the material to be tested using any one or a combination of KASP primer pairs selected from a specific primer set for molecular markers linked to wheat grain-related trait genes, wherein the specific primer set selected from molecular markers linked to wheat grain-related trait genes comprises the following KASP primer pairs: KASP-AX-108776765 Primer pairs ， The following primer pairs are included: Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTTGATTTCTAGAATAAAACGTTCCCATTCTG-3', forward primer B: 5'-GAAGGTCGGAGTCAACGGATTTGATTTCTAGAATAAAACGTTCCCATTCC-3', Reverse primer: 5′-GGCCCCTTACAAATACAGTATAAAACA-3′; KASP-AX-110419159 Primer pairs ， The following primers are included: Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTGCCAAACCGTGAGCCTAATAA-3', Forward primer B: 5'-GAAGGTCGGAGTCAACGGATTGCCAAACCGTGAGCCTAATAT-3', Reverse primer: 5′-AGTGCTGCTGTAATTCTTTCCC-3′; KASP-AX-110385388 Primer pairs ， The following primers are included: Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTCCGGTGAGGGTTTTAATCACATG-3', Forward primer B: 5'-GAAGGTCGGAGTCAACGGATTCCGGTGAGGGTTTTAATCACATC-3', Reverse primer: 5′-CCTTGCTGGCTATGACTACCC-3′; KASP-AX-110488530 Primer pairs ， The following primers are included: Forward primer A: 5'-GAAGGTGACCAAGTTCATGCTTCACCACTGGAGAAAACCGT-3', Forward primer B: 5'-GAAGGTCGGAGTCAACGGATTTCACCACTGGAGAAAACCGC-3', Reverse primer: 5′-CACCAGTGATGGCTTTACGTAAT-3′; For the KASP-AX-108776765 Amplification products of primer pairs ， If the amplified product shows blue fluorescence, the material to be tested carries genes for grain-related traits and has excellent average grain length, average grain width, average grain circumference, average grain area and 1000-grain weight traits; if the amplified product shows orange-yellow fluorescence, the material type to be tested does not carry genes for grain-related traits; For the KASP-AX-110419159 If the amplified product of the primer pair shows blue fluorescence, the material to be tested carries genes for grain-related traits and has excellent average grain length, average grain width, grain width-to-length ratio, average grain circumference, and average grain area traits; if the amplified product shows orange-yellow fluorescence, the material to be tested does not carry genes for grain-related traits; For the KASP-AX-110385388 If the amplification product of the primer pair shows orange-yellow fluorescence, the material to be tested carries genes for grain-related traits and has excellent grain width-to-length ratio, average grain circumference, average grain area, and 1000-grain weight traits. If the amplification product shows blue fluorescence, the material to be tested does not carry genes for grain-related traits. For the KASP-AX-110488530 If the amplification product of the primer pair shows orange-yellow fluorescence, the material to be tested carries genes for grain-related traits and has excellent average grain length, average grain width, grain width-to-length ratio, average grain circumference, average grain area and 1000-grain weight. If the amplification product shows blue fluorescence, the material to be tested does not carry genes for grain-related traits.

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