KASP primer set for detecting new grain weight loci overcoming pigm micro-grain effect and application thereof

Abstract

The application discloses a KASP primer group for detecting a new grain weight locus overcoming a Pigm small grain effect and application of the KASP primer group, the KASP marker primer comprises a forward primer F, a reverse primer R1 and a reverse primer R2, can effectively genotype a SNP site (C / T) at 24259747 of the 7th chromosome of rice, and can more efficiently screen and identify the to-be-tested material under laboratory conditions, and the like. By using the marker, a high-grain-weight allele is introduced into a rice blast resistant low-grain-weight line by a molecular marker assisted selection method, and the introduction line is improved in grain weight level and a new disease-resistant high-yield germplasm resource is created.

Description

Technical Field

[0001] This invention belongs to the field of crop genetics and breeding, and relates to a linked KASP marker that can overcome the Pigm small grain effect and its breeding application. Background Technology

[0002] Nearly 60% of my country's population relies on rice as their staple food, making it a major rice-consuming nation. With population growth and declining arable land, increasing rice production is directly related to national food security and social stability. Furthermore, in rice blast resistance breeding practices, high levels of resistance from disease-resistant genes are often accompanied by yield losses. Grain weight, as one of the three key factors determining rice yield, also affects the quality of rice and paddy, especially milling yield and processing quality. Although some important grain weight regulatory genes / QTLs have been cloned, only a few have been applied in breeding practices. Therefore, identifying new grain weight regulatory sites with breeding value is crucial for further increasing rice yield and has significant practical application value for ensuring safe rice production.

[0003] Rice blast is a devastating disease that seriously threatens the safe production of rice worldwide. Breeding practices have shown that using resistance genes to cultivate disease-resistant varieties is the most economical, effective, and environmentally friendly measure to control rice blast. Among the numerous resistance genes, the broad-spectrum and durable resistance locus Pigm in the Chinese local variety "Gumei 4" has long been used as an excellent source of resistance in rice blast resistance breeding. However, in the process of using the Pigm gene in breeding practices, the applicant team found that the grain weight and grain size of near isogenic lines (NILs) carrying the Pigm gene were significantly reduced in multiple japonica rice backgrounds, indicating that the small grain effect caused by Pigm is prevalent in japonica rice.

[0004] Single nucleotide polymorphisms (SNPs) mainly refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. Competitive allele-specific PCR (KASP) uses specific matching of primer terminal bases to genotype SNPs and detect InDels, exhibiting high stability and accuracy.

[0005] Currently, there are no reports on particle weight sites that can overcome the Pigm small particle effect. Summary of the Invention

[0006] To address the aforementioned issues, this application is the first to discover an SNP site associated with rice grain weight. The T variation at this site can overcome the small grain effect of Pigm. Furthermore, a KASP primer set was designed for this SNP site, and the SNP site was applied to rice breeding to accelerate the rice breeding process.

[0007] Specifically, this application is implemented through the following technical solution:

[0008] This application first provides a KASP primer set for detecting new particle repositioning sites that overcome the Pigm small particle effect. The primer set includes a forward primer F (nucleotide sequence shown in SEQ ID NO.1), a reverse primer R1 (nucleotide sequence shown in SEQ ID NO.2), and a reverse primer R2 (nucleotide sequence shown in SEQ ID NO.3). Primer R1 contains a FAM fluorescent group, and primer R2 contains a HEX fluorescent group.

[0009] F(SEQ ID NO.1):ATTACCTCATAGGAAGGACATGAAG;

[0010] R1 (SEQ ID NO.2): gaaggtgaccaagttcatgct GTGAAAACGGTGATTGGGAA;

[0011] R2 (SEQ ID NO.3): gaaggtcggagtcaacggatt GTGAAAACGGTGATTGGGAG.

[0012] This KASP primer set can detect the SNP locus located at position 24259747 on chromosome 7 of the rice genome (GenBank: AP014963.1). The genotype of this locus is C / T. Rice grains containing the T allele have a significantly higher grain weight than rice grains containing the C allele. Therefore, the KASP primer set can be used to distinguish between the two.

[0013] Secondly, this application provides the application of the above-mentioned KASP primer set in identifying rice grain weight. Specifically, the specific steps are as follows: using sample rice DNA as a template, the above-mentioned KASP primer set is used as primers for PCR amplification. After the reaction is completed, the fluorescence signal generated by each reaction well is collected, and the genotype of the molecular marker is determined according to the type of fluorescence signal: if only FAM fluorescence signal is detected, the genotype of the SNP site is T, and the rice sample to be tested contains the high grain weight gene qGW7; if only HEX fluorescence signal is detected, the genotype of the SNP site is C, and the rice sample to be tested does not contain the high grain weight gene qGW7; if both fluorescence signals are detected simultaneously, the rice sample to be tested is a heterozygous genotype; the grain weight of rice containing allele T is significantly higher than that of rice containing allele C; the rice is a rice line containing the Pigm site.

[0014] Furthermore, the above PCR amplification system consisted of: 2.5 μL of 20 ng / μL DNA, 5.0 μL of 2x KASP Master mix, 0.4 μL of forward primer F (10 μM), 0.15 μL of reverse primer R1 (10 μM), 0.15 μL of reverse primer R2 (10 μM), and 1.8 μL of ddH2O. The system was then placed in a 384-well PCR instrument for reaction.

[0015] PCR amplification program: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 58℃ for 60 s, 10 cycles (decreasing by 0.8℃ per cycle); 94℃ denaturation for 20 s, 57℃ annealing for 60 s, 32 cycles.

[0016] Third, this application provides the application of the above-mentioned KASP primer set in rice breeding, including the following steps:

[0017] ① Using rice DNA samples containing the Pigm locus as templates, PCR amplification was performed using the above-mentioned KASP primer set. After the reaction was completed, the fluorescence signal generated by each reaction well was collected, and the genotype of the molecular marker was determined according to the type of fluorescence signal: if only FAM fluorescence signal was detected, the genotype of the SNP locus was T, and it was determined to be a high-grain-weight line containing Pigm; if only HEX fluorescence signal was detected, the genotype of the SNP locus was C, and it was determined to be a low-grain-weight line containing Pigm.

[0018] Using a low-grain-weight line containing Pigm as the recipient and a high-grain-weight line containing Pigm as the donor, F1 generation seeds were obtained by hybridization.

[0019] ② Cultivate F1 seeds and backcross multiple generations using the recipient as the recurrent parent until BC3F1 is obtained. At the seedling stage of each generation, DNA samples are extracted and compared using the aforementioned KASP markers. Plants containing the high grain weight allele are selected for hybridization with the recipient parent.

[0020] ③ In the BC3F1 generation, seeds containing the high grain weight allele are harvested to obtain BC3F2 seeds;

[0021] ④ BC3F2 were planted separately, and DNA was extracted during the seedling stage. The KASP markers were used to screen for single plants with high grain weight homozygous alleles.

[0022] ⑤ The individual plants obtained in step ④ are continuously self-pollinated until the agronomic traits are stable to obtain the template line; the target line is a rice line that takes into account both high disease resistance (containing Pigm) and high grain weight.

[0023] In this application, the term "small grain effect" refers to the phenomenon that grain weight decreases and grains become smaller after the Pigm gene is introduced into different japonica rice backgrounds through multi-generation backcrossing during the breeding of rice blast resistance.

[0024] This application evaluated the grain weight of multiple background NILs (containing the broad-spectrum resistance gene Pigm) in japonica rice and ultimately identified a line with significantly increased grain weight in NILs_MGJ30 (Pigm). Furthermore, a novel grain weight-regulating SNP, qGW7, located at position 24259747 on chromosome 7 of the rice genome, was discovered through genome-wide association analysis. In one embodiment of this application, introducing this site into NIL_Yangjing687 (Pigm) significantly increased its grain weight, indicating that this site has important application value in high-yield and disease-resistant rice breeding.

[0025] This application designs a KASP primer set targeting this SNP locus, enabling rapid, accurate, efficient, and high-throughput detection of grain weight alleles in breeding materials at an early stage, eliminating the need for post-harvest seed testing. This promotes genetic improvement of high-grain-weight rice varieties and increases breeding efficiency. Furthermore, compared to existing molecular markers, KASP detection technology is simple to operate, offers high throughput at low cost, and provides accurate and reliable results. It also eliminates the need for environmentally polluting reagents such as EB or polyacrylamide, making it valuable for breeding high-yielding and disease-resistant varieties and promising for commercial breeding applications. Attached Figure Description

[0026] Figure 1 This is a graph showing the detection of small grain genetic effects of Pigm in the context of japonica rice in an example.

[0027] Among them, A: The grain weight of NILs carrying Pigm was significantly reduced in all 10 japonica rice backgrounds; B: The grain width and grain thickness of NILs carrying Pigm were smaller in the backgrounds of Wuyunjing 24, Yangjing 687, Hongguangjing 1 and Yangjing 5118.

[0028] Figure 2 Examples include photographs of rice seeds carrying Pigm-laden NILs against a background of multiple japonica rice varieties and statistical charts of grain weight detection results;

[0029] Wherein, A: is a schematic diagram of the grain weight statistics in NILs_MGJ30(Pigm), B: is a grain weight photo of NIL5-NIL12, and CF are schematic diagrams of the statistical results of plot yield, brown rice rate, milled rice rate and head rice rate of NIL5-NIL12 respectively.

[0030] Figure 3 Manhattan plot of the location of qGW7 site;

[0031] Figure 3It can be seen that there is a new grain weight locus qGW7 on chromosome 7 of rice at positions 24,181,760,24,515,093. There is an effective nucleotide difference between high-grain-weight lines and low-grain-weight lines at position 24,259,747 on chromosome 7, which is T / C.

[0032] Figure 4 This is a schematic diagram of the KASP primer set.

[0033] Figure 5 This is a fractal diagram of the chained KASP markers in Embodiment 1 of the present invention;

[0034] In this diagram, A (green dots) represents the distribution points of the high grain weight allele, B (blue dots) represents the distribution points of the low grain weight allele, C (red dots) represents the distribution points of the heterozygous genotype, and D (gray dots) represents the distribution points of the negative control.

[0035] Figure 6 Photos and statistical diagrams of grain weight for breeding high-grain-weight disease-resistant rice varieties;

[0036] Among them, A is a photo of rice seeds after qGW7 was introduced with NIL_Yangjing687(Pigm), which shows that the grain width and thickness increased; B and C are schematic diagrams of the thousand-grain weight and single-plant yield after qGW7 was introduced with NIL_Yangjing687(Pigm), which show that the thousand-grain weight and single-plant yield both increased after the introduction of this site. Detailed Implementation

[0037] The following embodiments are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all purchased from conventional biochemical reagent stores. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention.

[0038] This invention relates to the definitions of abbreviations and terms as follows:

[0039] KASP: Competitive allele-specific PCR.

[0040] SNP: Single nucleotide polymorphism.

[0041] NIL: near-isogenic line.

[0042] The varieties Gu Mei No. 4, Hua Jing No. 5, Yang Jing No. 687, Huai Dao No. 5, Xu Dao No. 9, Xu Dao No. 12, Wu Yun Jing No. 24, Nan Jing No. 9108, Hong Guang Jing No. 1, Si Dao No. 1167, Yang Jing No. 5118, and MGJ30 mentioned in the examples were all collected and stored by the applicant's laboratory.

[0043] Example 1 Mapping of qGW7, a novel grain weight locus that can overcome the small grain effect of Pigm

[0044] 1.1 Verification of the small grain effect caused by Pigm in japonica rice background

[0045] Using Gumei 4 (Pigm) as the donor, 10 japonica rice varieties including Huajing 5, Yangjing 687, Huaidao 5, Xudao 9, Xudao 12, Wuyunjing 24, Nanjing 9108, Hongguangjing 1, Sidao 1167 and Yangjing 5118 included in the China National Rice Data Center were used as recurrent parents respectively. Continuous backcrossing for 4 generations to BC4F1 was carried out in combination with molecular marker-assisted selection, followed by three generations of selfing. Finally, NILs with agronomic traits similar to the recurrent parents and homozygous Pigm gene were obtained, and the number of NILs per background was more than 25. The materials were planted at Wanfu Base of Jiangsu Lixiahe District Institute of Agricultural Sciences in 2019. After harvest at maturity, the grain weight and grain size of all individual plants were measured. The obtained results are shown in Figure 1 .

[0046] Figure 1 , in which A is a schematic diagram of the statistical results of average grain weight of corresponding Pigm-carrying NILs in 10 japonica rice backgrounds (the number of NILs per background is more than 25). It can be seen that the average 1000-grain weight of multiple NIL lines is significantly reduced; B is a grain photograph of Pigm NILs in the backgrounds of Wuyunjing 24, Yangjing 687, Hongguangjing 1 and Yangjing 5118. It can be seen that the grain width and grain thickness become smaller. Therefore, it is considered that the small grain effect caused by Pigm is widespread in japonica rice backgrounds.

[0047] 1.2 Excavation of Pigm high grain weight derived lines

[0048] In this example, Gumei 4 (Pigm) was further used as the donor, multiple japonica rice varieties were used as recurrent parents, continuous backcrossing for 4 generations to BC4F1 was carried out in combination with molecular marker-assisted selection, followed by three generations of selfing, and Pigm-carrying NILs in multiple japonica rice backgrounds were constructed (the number of NILs per background is more than 10). Among them, there are 12 Pigm-carrying NILs in the background of MGJ30 (an intermediate breeding material, preserved by Jiangsu Lixiahe District Institute of Agricultural Sciences), which were named NIL1-NIL12 (as shown in Figure 2 A in). Then the grain weight and grain size of NILs in different japonica rice backgrounds were detected according to the method disclosed in the literature "Copy number variation at the GL7 locus contributes to grain size diversity in rice", and finally the lines NIL5-NIL12 with significantly increased grain weight were found in NILs_MGJ30 (Pigm) (as shown in Figure 2As shown in Figure A), the corresponding grain size examination shows that the grain weight of NIL5-NIL12 increases with the increase of grain width and grain thickness (e.g., Figure 2 (As shown in B).

[0049] Increased grain weight often improves yield potential and rice milling rate, resulting in better economic benefits. This embodiment further uses the method disclosed in the literature "The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality" to detect plot yield, and the method disclosed in the national standard GB / T 5495—2008 "Grain and Oil Inspection: Rice Paddy Peel Yield Test" to detect hulling rate, milled rice rate, and head rice rate. Corresponding to the increase in grain weight, the plot yield of NIL5-NIL12 (e.g., Figure 2 As shown in C), roughness ratio (as shown in C) Figure 2 As shown in D), the rice milling rate (as shown in D) Figure 2 (as shown in E) and head rice rate (as shown in E) Figure 2 The values ​​of NIL5 and NIL12 (as shown in F) were significantly higher than those of the recurrent parent. Therefore, it is speculated that there is a particle weight regulation site in NIL5-NIL12 that can overcome the small particle effect of Pigm.

[0050] 1.3 Localization of the new granular repetitive site qGW7

[0051] To further pinpoint the effector sites, this embodiment constructed the high-grain-weight line NIL7 (i.e., Figure 2 The line with the highest grain weight shown in section A) and the low grain weight line NIL2 (i.e. Figure 2 The BC3F2 population (287 individuals) of the line with the lowest grain weight shown in A was used to perform whole-genome sequencing (sequencing depth 30×) on the high-grain-weight line NIL7 and the low-grain-weight line NIL2 and their F2 population.

[0052] Using the Nipponbare genome (IRGSP-1.0, https: / / rapdb.dna.affrc.go.jp) as the reference genome, sequence alignment was performed using BWA (http: / / bio-bwa.sourceforge.net) analysis software. The quality control parameters for SNP information extraction were set as follows: mapping quality value greater than 20, variation quality value greater than 50 for each site, and at least two reads supporting each base, with a MAF value > 0.05. The SNP extraction software used was GATK V4.1.4.1.

[0053] The thousand-grain weight data of all F2 individual plants, the high-grain-weight line NIL7, and the low-grain-weight line NIL2 were obtained and imported into Tassle (5.0) software along with the obtained material genotypes. Using a mixed linear model method, a new grain weight locus was located on rice chromosome 7. The applicant named this locus "qGW7". The Manhattan plot of the genome-wide association analysis of this locus is shown below. Figure 3 As shown.

[0054] Based on the physical location of the most significant SNP, the candidate region for qGW7 is approximately 333.3 kb between chromosome 7, 24,181,760..24,515,093. Further searching for mutation sites between the high-grain-weight line NIL7 and the low-grain-weight line NIL2 revealed a valid nucleotide difference of C / T at position 24,259,747, which was designed as a linked KASP marker (SNP site) for qGW7.

[0055] Based on the 300 bp sequences upstream and downstream of the aforementioned SNP site, KASP-labeled primers were designed using Primer Premier 5.0 software. These include forward primer F (nucleotide sequence shown in SEQ ID NO.1), reverse primer R1 (nucleotide sequence shown in SEQ ID NO.2), and reverse primer R2 (nucleotide sequence shown in SEQ ID NO.3), with specific sequences as follows: Figure 4 As shown.

[0056] Figure 4 In the sequence, the underlined part of primer R1 is the FAM universal fluorescent tag sequence, and the underlined part of primer R2 is the HEX universal fluorescent tag sequence.

[0057] The KASP markers described above can be used for high-throughput detection of the qGW7 locus in rice materials: if only the FAM fluorescence signal corresponding to R1 is detected in the PCR product, the detection site base is T, indicating that the test material contains the high grain weight allele; if only the HEX fluorescence signal corresponding to R2 is detected, the detection site base is C, indicating that the test material contains the low grain weight allele; if both FAM and HEX fluorescence signals are detected simultaneously, the test material is heterozygous. Rice containing the T allele has a significantly higher grain weight than rice containing the C allele.

[0058] Example 2: Detection of Linked KASP Markers

[0059] Validation was performed on individual plants from the NIL1-NIL12 and BC3F2 populations. Genomic DNA was extracted from individual plant leaves using the CTAB method and amplified by PCR using the primer set described above, in a 10 μL reaction volume.

[0060] The reaction system consisted of: 2.5 μL of 20 ng / μL DNA, 5.0 μL of 2x KASP Master mix, 0.7 μL of KASP Assay mix (upstream and downstream primer mixture), and 1.8 μL of ddH2O. The system was placed in a 384-well PCR instrument for the reaction.

[0061] The KASP Assay mix (upstream and downstream primer mixture) is prepared as follows: 0.4 μL of F primer (10 μM), 0.15 μL of R1 primer (10 μM), and 0.15 μL of R2 primer (10 μM).

[0062] PCR amplification program: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 58℃ for 60 s, 10 cycles (decreasing by 0.8℃ per cycle); 94℃ denaturation for 20 s, 57℃ annealing for 60 s, 32 cycles.

[0063] After the cycle was completed, the results were detected in a 384-well real-time PCR instrument. Figure 5 As shown. In Figure 5 In the diagram, the X-axis represents the HEX fluorescence signal coordinate axis, and the Y-axis represents the FAM fluorescence signal coordinate axis. Figure 5 In the diagram, A (green dot) represents the distribution point of the high grain weight allele, B (blue dot) represents the distribution point of the low grain weight allele, C (red dot) represents the distribution point of the heterozygous genotype, and D (gray dot) represents the distribution point of the negative control. This demonstrates that the marker can accurately distinguish between the high grain weight allele, the low grain weight allele, and the heterozygous genotype.

[0064] The detection results of NIL1-NIL12 and some BC3F2 populations (1-20) are shown in Table 1. In Table 1, NIL1-NIL4 are low-grain-weight lines, corresponding to the HEX fluorescence signal, with C at bases 24,259,747; NIL5-NIL12 are high-grain-weight lines, corresponding to the FAM fluorescence signal, with T at bases 24,259,747. In addition, in the BC3F2 population, high-grain-weight plants have T at bases 24,259,747; low-grain-weight plants have C at bases 24,259,747; and plants with a thousand-grain weight between 27-28g correspond to the FAMHEX fluorescence signal, with a T:C heterozygous genotype at bases 24,259,747.

[0065] Table 1. Detection results of NIL1-NIL12 and some BC3F2 groups (1-20).

[0066]

[0067] Example 3: A method for breeding highly disease-resistant, high-grain-weight rice varieties

[0068] This embodiment provides a method for cultivating high-grain-weight disease-resistant rice lines based on the KASP primer set of Example 1, including the following steps:

[0069] ① Rice varieties containing the Pigm locus (in this example, NIL_Yangjing687 (Pigm) is used, which is a previously obtained line with high resistance to rice blast but reduced grain weight, such as...) Figure 1 As shown in Figure A), it was used as the female parent and synergized with the highly resistant rice blast high-grain-weight line NIL7 (… Figure 2 (As shown in A) 30 F1 seeds were obtained through hybridization;

[0070] ② Cultivate F1 seeds and obtain 40 BC1F1 seeds by crossing with NIL_Yangjing687(Pigm);

[0071] ③ Cultivate BC1F1 seeds, extract DNA samples from seedling plants, and compare and detect them using the KASP primer set obtained in Example 1. Select individual plants containing the high grain weight allele (i.e., T gene selected using the above KASP primers) and with agronomic traits similar to NIL_Yangjing687(Pigm) and cross them with NIL_Yangjing687(Pigm) to obtain 40 BC2F1 seeds.

[0072] ④ Following the method in step ③, 150 BC3F1 seeds were finally obtained;

[0073] ⑤ Cultivate BC3F1 seeds, extract DNA samples from seedling plants, use the above-mentioned KASP markers for comparison and detection, screen to obtain single plants carrying the high grain weight allele, and finally obtain 6 BC3F1 line progeny through agronomic trait screening.

[0074] ⑥ Twenty plants were planted for each line, and the qGW7 genotype was tested. One homozygous plant with excellent agronomic traits was selected from each line, resulting in five BC3F3 lines. Field yield comparisons were conducted on each line, and five lines (named Line 1-5) with agronomic traits essentially identical to the recurrent parents were ultimately obtained. Results showed that Line 1-5, carrying the qGW7 high-grain-weight allele, had significantly higher grain width and thickness than Yangjing 687 (e.g., ...). Figure 6 As shown in Figure A), the weight of a thousand grains (as shown in Figure A) Figure 6 (as shown in B) and yield per plant (as shown in B) Figure 6 The results (shown in C) also show a significant improvement. It is evident that the KASP primer set obtained in Example 1 can effectively accelerate the breeding process and screen rice lines that balance disease resistance (Pigm site) and grain weight.

[0075] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. sequence list <110> Jiangsu Lixiahe Area Agricultural Science Research Institute <120> KASP primer set for detecting new particle reposition sites that overcome Pigm small particle effect and its application <141> 2021-12-31 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <400> 1 attacctcat aggaaggaca tgaag 25 <210> 2 <211> 41 <212> DNA <213> Artificial Sequence <400> 2 gaaggtgacc aagttcatgc tgtgaaaacg gtgattggga a 41 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <400> 3 gaaggtcgga gtcaacggat tgtgaaaacg gtgattggga g 41

Claims

1. The application of the KASP primer set in the determination of rice grain weight, characterized in that, The KASP primer set consists of forward primer F (nucleotide sequence as shown in SEQ ID NO.1), reverse primer R1 (nucleotide sequence as shown in SEQ ID NO.2), and reverse primer R2 (nucleotide sequence as shown in SEQ ID NO.3). The application refers to using rice DNA as a template and the KASP primer set as primers for PCR amplification and collection of fluorescence signals. If only FAM fluorescence signal is detected, the rice sample is determined to contain allele T; if only HEX fluorescence signal is detected, the rice sample is determined to contain allele C; the grain weight of rice containing allele T is significantly higher than that of rice containing allele C.

2. The application as described in claim 1, characterized in that, The PCR amplification refers to: PCR amplification system: 2.5 μL of 20 ng / μL sample DNA, 5.0 μL of 2 x KASP Master mix, 0.4 μL of 10 μM forward primer F, 0.15 μL of 10 μM reverse primer R1, 0.15 μL of 10 μM reverse primer R2, and 1.8 μL of ddH2O; PCR amplification program: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 58℃ for 60 s, 10 cycles, decreasing by 0.8℃ per cycle; 94℃ denaturation for 20 s, 57℃ annealing for 60 s, 32 cycles.

3. The application of KASP primer sets in rice breeding is characterized by... The KASP primer set consists of forward primer F (nucleotide sequence as shown in SEQ ID NO.1), reverse primer R1 (nucleotide sequence as shown in SEQ ID NO.2), and reverse primer R2 (nucleotide sequence as shown in SEQ ID NO.3); the rice breeding refers to the identification of rice grain weight, and the steps are as follows: 1) Using rice DNA containing the Pigm site as a template, PCR amplification was performed using the KASP primer set as primers. After the reaction was completed, the fluorescence signal was collected. If only the FAM fluorescence signal was detected, the rice sample was determined to contain the allele T, and the corresponding sample was a high-grain-weight rice variety. If only HEX fluorescence signal is detected, the rice sample is determined to contain allele C, and the corresponding sample is a low grain weight rice variety. Using low-grain-weight rice varieties as recipients and high-grain-weight rice varieties as donors, F1 generation seeds were obtained through hybridization. 2) Cultivate F1 seeds and backcross them repeatedly with the recipient as the recurrent parent until BC3F1 is obtained; DNA samples were extracted from the seedlings of each generation, and step 1) was repeated to screen high-grain-weight rice varieties for hybridization with the recipient parent. 3) Harvest the seeds of high-grain-weight rice varieties in the BC3F1 generation to obtain BC3F2 seeds; 4) Plant BC3F2 lines separately, extract DNA during the seedling stage, repeat step 1) to screen for high grain weight rice lines, which are single plants with high grain weight homozygous alleles. 5) The single plants obtained in step 4) are continuously self-pollinated until the agronomic traits are stable, thus obtaining a rice variety that combines high disease resistance and high grain weight.

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