Rice grain shattering site qSH10 and its molecular markers and applications
By designing KASP primers at the qSH10 site at 17843900bp on rice chromosome 10, the problem of inefficiently selecting rice with moderate grain shattering in traditional breeding methods was solved. This enabled rapid and accurate identification and breeding of rice grain shattering, shortened the breeding cycle, and reduced costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INSTITUTE OF CROP SCIENCE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
- Filing Date
- 2026-03-20
- Publication Date
- 2026-07-31
AI Technical Summary
Existing technologies make it difficult to efficiently and accurately breed rice varieties with moderate grain shattering and excellent overall traits. Traditional breeding methods are time-consuming and costly.
By identifying the qSH10 site at 17843900bp on rice chromosome 10, KASP primers were designed for molecular marker detection. PCR amplification was performed using KASP primer mix, and combined with nucleotide allelic variation-specific and universal primers, efficient identification and selection of rice grain shattering was achieved.
It enables rapid and accurate identification of rice grain shattering, significantly shortens the breeding cycle, reduces field screening costs, and cultivates new varieties that meet the needs of modern production.
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Figure CN121874394B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to agricultural biotechnology engineering, specifically involving the rice grain shattering site qSH10 and its molecular markers and applications. Background Technology
[0002] Rice shattering is a crucial agronomic trait affecting harvest efficiency and yield, and a key target trait for selection during domestication and breeding. Wild rice typically exhibits strong shattering to facilitate natural seed dispersal, but in cultivated rice, excessive shattering can lead to grain detachment at maturity, resulting in yield loss. Therefore, appropriately reducing shattering and increasing grain settling strength has become an important direction in modern rice breeding.
[0003] Rice shattering is a complex quantitative trait regulated by multiple genes. Conventional breeding methods relying on traditional phenotypic selection are insufficient for efficiently and accurately breeding varieties with moderate shattering and excellent overall traits. Identifying the key gene loci regulating rice shattering and developing molecular markers based on this understanding, through molecular design breeding techniques, can achieve multi-gene aggregation breeding, avoiding the randomness of gene recombination in traditional hybridization, significantly shortening the breeding cycle, and reducing field screening costs. This allows for the efficient and precise breeding of new rice varieties with suitable shattering that meet the needs of modern production. Summary of the Invention
[0004] Through research, this invention discovered a site qSH10 at 17843900bp on chromosome 10 of rice that is significantly associated with grain shattering. It was found that the grain shattering of germplasm carrying T nucleotide allelic variations was significantly stronger than that of germplasm carrying C nucleotide allelic variations, thus completing this invention.
[0005] This invention provides a method for detecting molecular markers of rice grain shattering. The molecular marker of rice grain shattering is qSH10, a site located at 17843900 bp on chromosome 10 that is significantly associated with grain shattering. Specifically, it is the nucleotide difference at the physical location of 17843900 bp. Furthermore, the grain shattering of germplasm carrying T nucleotide allelic variation at this site is significantly stronger than that of germplasm carrying C nucleotide allelic variation.
[0006] KASP primers were used for detection.
[0007] Specifically, the KASP primers include strong schistosomiasis-specific primers for detecting T nucleotide allelic variant sites, weak schistosomiasis-specific primers for detecting C nucleotide allelic variant sites, and universal primers.
[0008] More specifically, the specific primer for detecting strong leptogranulation allelic variants carrying T nucleotide allelic variant sites is: 5'-GAAGGTCGGAGTCAACGGATTCTACCAATCTCTATGGTGGCACA-3' (SEQ ID No. 1).
[0009] The specific primer for detecting weak leptogranulation allelic variants carrying C nucleotide allelic variants is: 5'-GAAGGTGACCAAGTTCATGCTTACCAATCTCTATGGTGGCACG-3' (SEQ ID No. 2).
[0010] The universal primer is: 5'-TGTGCAATCTGCGGTGGCACTC-3' (SEQ ID No. 3).
[0011] Preferably, the detection method is gene sequencing or molecular amplification.
[0012] The detection method, preferably, is molecular amplification, and the method comprises the following steps: extracting genomic DNA from the rice to be tested, using the genomic DNA as a template, and performing PCR amplification using the KASP primers to obtain PCR products.
[0013] Specifically, in the PCR amplification system, the concentrations of the strong chafing allelic variant-specific primers for detecting T nucleotide allelic variant sites and the weak chafing allelic variant-specific primers for detecting C nucleotide allelic variant sites are 30-45 μmol / μL, and the concentration of the universal primers is 80-100 μmol / μL. The three primers are mixed in a volume ratio of 1:1:1 to form the KASP primer mix. The total reaction volume is 10.14 μL, and the reaction system includes: 5 μL of DNA, 5 μL of 2xKASP Master Mix, and 0.14 μL of KASP primer mix.
[0014] The amplification program was as follows: (1) 94℃ pre-denaturation for 15 min; (2) 94℃ denaturation for 20 s, 61℃ extension for 60 s, decreasing at a rate of 0.6℃ / cycle, for 10 cycles; (3) 94℃ denaturation for 20 s, 55℃ extension for 60 s, for 26 cycles.
[0015] The present invention also provides the application of the method for detecting the molecular marker of rice shattering in identifying rice shattering or cultivating rice with target shattering.
[0016] Specifically, the application is in the identification of rice grain shattering. The molecular marker detection method for rice grain shattering is used to obtain the results of the molecular marker to determine the rice grain shattering. The grain shattering of germplasm carrying T nucleotide allelic variation at this site is significantly stronger than that of germplasm carrying C nucleotide allelic variation.
[0017] Specifically, the application is in the cultivation of rice with target grain shattering. The detection method described above is used to assist in judging the grain shattering of rice plants during the rice cultivation process. Selection is made according to the requirements of the target grain shattering. Among them, the grain shattering of germplasm carrying T nucleotide allelic variation at this site is significantly stronger than that of germplasm carrying C nucleotide allelic variation.
[0018] The application further includes the following detection steps: identifying the grain-shattering property of the rice to be tested based on the nucleotide sequence of the PCR product: if the PCR product is classified as T-type, the rice to be tested is or is a candidate rice variety with strong grain-shattering property; if the PCR product is classified as C-type, the rice to be tested is or is a candidate rice variety with weak grain-shattering property.
[0019] The method for identifying rice grain shattering in this invention has been verified to be accurate and reliable. It can achieve rapid batch identification of large-scale rice materials, meet the practical needs of high-throughput screening in breeding work, and has application value. Attached Figure Description
[0020] Figure 1 The results of genome-wide association analysis (GWAS) of grain loss rate in 314 rice accessions are shown in the figure. qSH10 in this invention is marked with an arrow.
[0021] Figure 2 Comparison of grain loss rate between two allelic variants of qSH10.
[0022] Figure 3 : genotyping results of qSH10 specific molecular markers.
[0023] Figure 4 : Validation of the effectiveness of qSH10 in the identification of grain shedding. Detailed Implementation
[0024] Example 1: Identification of qSH10, a rice grain-shattering site, and its specific molecular marker.
[0025] I. Identification of qSH10, a locus for grain shattering in rice
[0026] 1. Test materials and phenotypic identification
[0027] We used 314 rice germplasm accessions provided by the Chinese Academy of Agricultural Sciences to identify rice grain shattering using the free-fall method. At maturity, when the grains were firm and over 80% of the spikelets were mature, the air-dried rice panicles were placed at a height of 1.5m and allowed to fall naturally onto a ground supported by an iron plate. This process was repeated three times. The percentage of shattered spikelets out of the total number of spikelets (including filled grains, empty grains, and shattered grains) was calculated.
[0028] 2. Genome-wide association analysis of rice grain shattering
[0029] Genome-wide association analysis (GWAS) was performed using 3,258,526 SNPs from 314 rice whole genome accessions and grain shattering rate data. GWAS was conducted using the Tassel (v5) software package (Reference: Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ramdoss Y, Buckler ES. (2007) TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics 23:2633-2635.), and was calculated using a mix linear model (MLM) in conjunction with the kinship matrix and population structure Q matrix. P < 10 was considered a threshold. -4 As a significance threshold, a site qSH10, significantly associated with shattering, was identified at 17,843,900 bp on chromosome 10. Figure 1 ).
[0030] 3. Allelic variation analysis at the qSH10 locus
[0031] To examine the correlation between qSH10 and shattering rate, this study compared the shattering rate of germplasm carrying different allelic variations at this locus. It was found that the shattering rate of germplasm carrying T nucleotide allelic variations was significantly higher than that of germplasm carrying C nucleotide allelic variations. Figure 2 (p < 0.001).
[0032] II. Development of qSH10-specific molecular markers
[0033] 1. Design of KASP primers
[0034] Based on the nucleotide differences of qSH10 at its physical location of 17,843,900 bp, a reference genome of ZS97 was extracted from the Ensembl genome database (http: / / ftp.ensemblgenomes.org / pub / release59 / plants / fasta / oryza_sativa_zs97 / ). Sequences within 100 bp above and below 17,843,900 bp were obtained using SnapGene software. Specific KASP markers for qSH10 were designed using Primer3Plus (https: / / www.primer3plus.com / ), including a strong shattering allelic variation-specific primer qSH10(T), a weak shattering allelic variation-specific primer qSH10(C), and a universal primer qSH10.
[0035] The primer sequence for the molecular marker qSH10(T) is: 5'-GAAGGTCGGAGTCAACGGATTCTACCAATCTCTATGGTGGCACA-3' (SEQ ID No. 1).
[0036] qSH10 (C) primer sequence: 5'-GAAGGTGACCAAGTTCATGCTTACCAATCTCTATGGTGGCACG-3' (SEQ ID No. 2).
[0037] qSH10 (Universal) primer sequence: 5'-TGTGCAATCTGCGGTGGCACTC-3' (SEQ ID No. 3).
[0038] Example 2: Method for identifying rice grain shattering
[0039] 1. Method for identifying the grain-shattering property of rice samples
[0040] Specifically, the following steps were taken: Genomic DNA was extracted from the rice to be tested, and molecular markers were used to amplify the PCR product using the genomic DNA as a template.
[0041] In the above experiment, the DNA extraction method steps are as follows: (1) Put a small amount of fresh rice leaves into a 2mL centrifuge tube containing steel balls, immerse it in liquid nitrogen for 10min, and then quickly grind it into powder using a sampler; (2) Add 600μL of CTAB buffer and bathe in a 65℃ water bath for 30min; (3) Add an equal volume of chloroform:isoamyl alcohol (24:1) solution and shake vigorously to mix thoroughly; (4) Centrifuge at 12,000rpm for 10min and aspirate 400μL of the supernatant into a new 1.5mL centrifuge tube; (5) Add 400μL of pre-cooled isopropanol and place it in a -20℃ refrigerator for at least 20min to precipitate DNA; (6) Centrifuge at 12,000rpm for 10min and discard the supernatant; (7) Air dry at room temperature and add 400μL of double-distilled sterile water (ddH2O) to dissolve the DNA.
[0042] Preparation of molecular marker primers: (1) The primers were purified by ULTRPAGE; (2) The primer powder was dissolved, and the concentrations of qSH10(T) and qSH10(C) were 36 μmol / μL, and the concentration of qSH10(Universal) was 90 μmol / μL; (3) The three primers were then mixed in a volume ratio of 1:1:1 to form KASP primer Mix.
[0043] The KASP genotyping experiment was performed as follows: 96-well plates were used for the reaction and instrumentation. The total reaction volume for each well was 10.14 μL. The reaction system included: 5 μL DNA, 5 μL 2x KASP Master Mix, and 0.14 μL KASP primer Mix. After completion, the 96-well plates were sealed with a centrifuged and fluorescently transparent membrane. After confirming that there were no air-permeable wells, PCR amplification was performed. Amplification program: (1) 94℃ pre-denaturation for 15 min; (2) 94℃ denaturation for 20 s, 61℃ extension for 60 s, decreasing at a rate of 0.6℃ / cycle, for 10 cycles; (3) 94℃ denaturation for 20 s, 55℃ extension for 60 s, for 26 cycles; (4) Data reading and analysis.
[0044] 2. Identify the grain-shattering property of the rice sample based on the nucleotide sequence of the PCR product:
[0045] If the PCR product is classified as T-type, then the rice being tested is or is a candidate rice variety with strong grain-shattering tendency.
[0046] If the PCR product is classified as type C, then the rice being tested is or is a candidate rice variety with weak grain shattering tendency.
[0047] Example 3: Application of qSH10 specific molecular marker in the identification of grain shattering in rice varieties
[0048] I. Test Materials
[0049] The tested materials were Yuzhuxiang, Zhenhui 084, Ganwanxian 31, Ganzaoxian 15, Ganzaoxian 37, Ganzaoxian 51, Z91-43, Jiayu 293, Jiazao 12, Zhong 106, Zhongyouzao 81, Guihuazhan, Jiayu 253, Jiayu 948, Xiangzaoxian 6, Erjiufeng, Shuanggui 36, Guangjie 9, Minhuazhan, Xinguangmei, Guiyefeng, Xiang'aizao 4, Xiangwanxian 13, Xiangzaoxian 3, Xing 2, Ganzaoxian 25, Ganzaoxian 34, Zhongyouzao 3, Guiyang'ai 49, Meiyazhan, Moliruanzhan, Feng'aizhan, Xiang'aizao 9, Feng'aizhan 1, and Liyuanzhan 2.
[0050] II. Rice grain shattering test
[0051] The particle shedding property of the test material was determined according to the method in step 1 of Example 1.
[0052] The results are shown in Table 1. The table shows that among the 35 rice varieties, the average grain loss rate was 4.96% for the following varieties: Yuzhuxiang, Zhenhui 084, Ganwanxian 31, Ganzaoxian 15, Ganzaoxian 37, Ganzaoxian 51, Z91-43, Jiayu 293, Jiazao 12, Zhong 106, Zhongyouzao 81, Guihuazhan, Jiayu 253, Jiayu 948, Xiangzaoxian 6, and Erjiufeng. The average shattering rate of the following rice varieties was 60.76%, all of which were rice varieties with relatively weak shattering. The average shattering rate of the following rice varieties was 60.76%, all of which were rice varieties with relatively strong shattering.
[0053] Table 1. Genotyping and grain-shattering characteristics of the tested rice varieties
[0054]
[0055]
[0056] III. Genotyping
[0057] Genomic DNA was extracted from the test material in step one. Using the obtained genomic DNA as a template, genotyping was performed using the qSH10 specific molecular marker. The specific experimental procedure is the same as step 1 in Example 2.
[0058] Allelic variants at the physical location 17843900 bp on chromosome 10 were detected using the qSH10 specific molecular marker. The results are shown in Table 1 and [Table data would be inserted here]. Figure 3As shown, the PCR products of Yuzhuxiang, Zhenhui 084, Ganwanxian 31, Ganzaoxian 15, Ganzaoxian 37, Ganzaoxian 51, Z91-43, Jiayu 293, Jiazao 12, Zhong 106, Zhongyouzao 81, Guihuazhan, Jiayu 253, Jiayu 948, Xiangzaoxian 6, and Erjiufeng were all classified as type C. According to the method for identifying rice grain shattering in Example 2, Yuzhuxiang, Zhenhui 084, Ganwanxian 31, Ganzaoxian 15, Ganzaoxian 37, Ganzaoxian 51, Z91-43, Jiayu 293, Jiazao 12, Zhong 106, Zhongyouzao 81, Guihuazhan, Jiayu 253, Jiayu 948, Xiangzaoxian 6, and Erjiufeng were all rice varieties with relatively weak grain shattering. Shuanggui 36 and Guangjie 9... The PCR products of the following rice varieties were all T-type: Shuanggui 36, Guangjie 9, Minhuazhan, Xinguangmei, Guiyefeng, Xiang'ai 4, Xiangwanxian 13, Xiangzaoxian 3, Xing 2, Ganzaoxian 25, Ganzaoxian 34, Zhongyouzao 3, Guiyang'ai 49, Meiyazhan, Moliruanzhan, Feng'aizhan, Xiang'ai 9, Feng'aizhan 1, and Liyuanzhan 2. They were identified according to the rice grain shattering identification method in Example 2. All of these varieties showed strong grain shattering.
[0059] It can be seen that the method for identifying rice grain shattering in this invention is completely consistent with the results of grain shattering identification in step two, and the average grain shattering rate of rice varieties with allelic variation T is 60.76%, which is significantly stronger than that of rice varieties with allelic variation C (average grain shattering rate 4.96%). Figure 4 (p < 0.001). This demonstrates that the method for identifying rice grain shattering in this invention is accurate and reliable.
[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for detecting a rice shattering molecular marker, characterized in that, The rice shattering molecular markers showed that germplasm carrying T nucleotide allelic variations exhibited significantly stronger shattering characteristics than germplasm carrying C nucleotide allelic variations. Detection was performed using KASP primers, which included a strong chafing-specific primer for detecting T nucleotide allelic variants, a weak chafing-specific primer for detecting C nucleotide allelic variants, and a universal primer; wherein, The specific primer for detecting strong leptomorphic allelic variants carrying T nucleotide allelic variant sites is: 5'-GAAGGTCGGAGTCAACGGATTCTACCAATCTCTATGGTGGCACA-3', The specific primers for detecting weakly deciduous allelic variants carrying C nucleotide allelic variations are: 5'-GAAGGTGACCAAGTTCATGCTTACCAATCTCTATGGTGGCACG-3', The universal primer is: 5'-TGTGCAATCTGCGGTGGCACTC-3'; The detection method is molecular amplification, and the steps are as follows: extract genomic DNA from the rice to be tested, use the genomic DNA as a template, perform PCR amplification using the KASP primers to obtain PCR products, and detect them. If the PCR product is classified as T-type, the rice to be tested is or is a candidate rice variety with strong grain shattering; if the PCR product is classified as C-type, the rice to be tested is or is a candidate rice variety with weak grain shattering.
2. The detection method as described in claim 1, characterized in that: In the PCR amplification system, the concentrations of the strong chafing allelic variant-specific primers for detecting T nucleotide allelic variants and the weak chafing allelic variant-specific primers for detecting C nucleotide allelic variants are 30-45 μmol / μL, and the concentration of the universal primers is 80-100 μmol / μL. The three primers are mixed in a volume ratio of 1:1:1 to form the KASP primer mix. The total reaction volume is 10.14 μL, and the reaction system includes: 5 μL DNA, 5 μL 2x KASP Master Mix, and 0.14 μL KASP primer mix. The amplification program was as follows: (1) 94℃ pre-denaturation for 15 min; (2) 94℃ denaturation for 20 s, 61℃ extension for 60 s, decreasing at a rate of 0.6℃ / cycle, for 10 cycles; (3) 94℃ denaturation for 20 s, 55℃ extension for 60 s, for 26 cycles.
3. The application of the detection method as described in any one of claims 1-2 in identifying rice grain shattering or cultivating rice with target grain shattering.
4. The application as described in claim 3, characterized in that, Its application in identifying rice grain shattering is to use the detection method described above to obtain the results of the molecular marker to determine rice grain shattering, wherein the grain shattering of germplasm carrying the T nucleotide allelic variation at this site is significantly stronger than that of germplasm carrying the C nucleotide allelic variation.
5. The application as described in claim 3, characterized in that, Its application is in the cultivation of rice with target grain shattering. The detection method described above is used to assist in judging the grain shattering of rice plants during the rice cultivation process. Selection is made according to the requirements of the target grain shattering. Among them, the grain shattering of germplasm carrying T nucleotide allelic variation at this site is significantly stronger than that of germplasm carrying C nucleotide allelic variation.