Rice cold tolerance site qcts4 at seedling stage and molecular marker and application thereof
By developing the seedling cold tolerance molecular marker qCTS4 at 32446985 bp on rice chromosome 4, and using KASP primers for PCR amplification and gene sequencing, the problems of long cycle and susceptibility to environmental interference in rice seedling cold tolerance identification in traditional breeding methods have been solved, achieving efficient and accurate cold tolerance identification and breeding assistance.
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
- 2026-03-27
- Publication Date
- 2026-08-04
AI Technical Summary
Traditional breeding methods are time-consuming and easily affected by environmental factors in the identification of cold tolerance in rice seedlings, which cannot meet the urgent need for cold-resistant varieties in rice production. Therefore, precise molecular tools are urgently needed to assist breeding.
A seedling cold tolerance molecular marker qCTS4 located at 32446985 bp on rice chromosome 4 was developed. KASP primers were used for detection, and rice cold tolerance was identified by PCR amplification and gene sequencing. Specific primers and universal primers were used to identify nucleotide allelic variations.
It enables efficient and accurate identification of cold tolerance in rice seedlings, significantly improving identification efficiency and accuracy, and can assist in the cultivation of cold-resistant rice varieties.
Smart Images
Figure CN121931285B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to agricultural biotechnology engineering, specifically involving the cold tolerance site qCTS4 in rice seedlings and its molecular markers and applications. Background Technology
[0002] Rice is a warm-season crop, and its seedlings are sensitive to low temperatures. Low temperatures can lead to decreased germination rates, yellowing and seedling death, stunted growth, and even death, severely impacting yield and planting area. Therefore, cultivating cold-resistant varieties can directly reduce losses caused by chilling injury and solidify the foundation for yield. Furthermore, it can expand rice planting areas, such as temperate zones, high-altitude regions, and northern rice-growing areas, optimizing global and national planting patterns and increasing overall production potential.
[0003] Cold tolerance in rice seedlings is a complex quantitative trait, regulated by multiple genes / QTLs and influenced by environmental conditions. Traditional breeding methods, including phenotypic screening, are time-consuming and susceptible to environmental interference, failing to meet the urgent need for cold-tolerant varieties in rice production. Therefore, precision molecular tools are urgently needed to assist breeding. This research aims to elucidate the genetic basis of cold tolerance in rice seedlings, develop relevant molecular markers for precision-assisted breeding, and establish a complete technical system from gene mapping to marker development and breeding application. This will promote a shift in rice cold-tolerant breeding from "experience-driven" to "precision design." Summary of the Invention
[0004] Through research, this invention discovered a site qCTS4 at 32446985 bp on rice chromosome 4 that is significantly associated with seedling cold tolerance. It was found that germplasm carrying A nucleotide allelic variants exhibited significantly stronger seedling cold tolerance than germplasm carrying G nucleotide allelic variants, thus completing this invention.
[0005] This invention provides a method for detecting molecular markers of cold tolerance in rice seedlings. The molecular marker for cold tolerance in rice seedlings is qCTS4, a site located at 32,446,985 bp on chromosome 4 that is significantly associated with cold tolerance in seedlings. Specifically, it refers to the nucleotide difference at the physical location of 32,446,985 bp. Furthermore, the seedling cold tolerance of germplasm carrying the A nucleotide allelic variation at this site is significantly stronger than that of germplasm carrying the G nucleotide allelic variation.
[0006] KASP primers were used for detection.
[0007] Specifically, the KASP primers include seedling-specific primers for detecting seedling-specific allelic variants carrying A nucleotide allelic variant sites, seedling-specific primers for detecting seedling-specific allelic variants carrying G nucleotide allelic variant sites, and universal primers.
[0008] More specifically, the seedling-specific primer for detecting seedling-stage cold-resistant allelic variants carrying A nucleotide allelic variant sites is: 5'-GAAGGTGACCAAGTTCATGCTCTATCAGGTGTAGGGCCCAGA-3' (SEQ ID No. 1).
[0009] The specific primer used to detect seedling-stage weak cold-resistant allelic variants carrying G nucleotide allelic variants is: 5'-GAAGGTCGGAGTCAACGGATTTATCAGGTGTAGGGCCCAGG-3' (SEQ ID No. 2).
[0010] The universal primer is: 5'-AGGGGTGGAGCCAGGTGTGG-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 seedling-stage cold-resistant allelic specific primers for detecting A nucleotide allelic variants and the seedling-stage weakly cold-resistant allelic variants for detecting G 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.
[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] Furthermore, the detection steps include: identifying the seedling cold tolerance of the rice to be tested based on the nucleotide sequence of the PCR product: if the PCR product is type A, then the rice to be tested is or is a candidate rice variety with strong seedling cold tolerance; if the PCR product is type G, then the rice to be tested is or is a candidate rice variety with weak seedling cold tolerance.
[0016] The present invention also provides the application of the detection method described herein in identifying the cold resistance of rice seedlings or in cultivating cold-resistant rice seedlings.
[0017] Specifically, the application is used to identify the cold tolerance of rice seedlings. The molecular marker results obtained by the detection method are used to determine the cold tolerance of rice seedlings. Among them, the seedling cold tolerance of germplasm carrying the A nucleotide allelic variant at the site is significantly stronger than that of germplasm carrying the G nucleotide allelic variant.
[0018] Specifically, the application is in the cultivation of cold-resistant rice seedlings. The detection method described above is used to assist in judging the cold resistance of rice seedlings during the rice cultivation process, and to select rice varieties with strong cold resistance during the seedling stage. Among them, the seedling cold resistance of germplasm carrying the A nucleotide allelic variation at this site is significantly stronger than that of germplasm carrying the G nucleotide allelic variation.
[0019] The application further includes the following detection steps: identifying the seedling cold tolerance of the rice to be tested based on the nucleotide sequence of the PCR product: if the PCR product is type A, then the rice to be tested is or is a candidate rice variety with strong seedling cold tolerance; if the PCR product is type G, then the rice to be tested is or is a candidate rice variety with weak seedling cold tolerance.
[0020] The method for identifying cold resistance in rice seedlings according to the present invention has been verified to have strong repeatability and high accuracy, which can greatly improve the identification efficiency and has wide application value. Attached Figure Description
[0021] Figure 1 Results of genome-wide association analysis (GWAS) of 372 rice accessions. Figures A and B show the Manhattan plot and QQ plot of the GWAS results for seedling cold tolerance, respectively. qCTS4 in this invention is indicated by an arrow in the figures.
[0022] Figure 2 Comparison of seedling cold tolerance levels between two allelic variants of qCTS4.
[0023] Figure 3 : genotyping results of specific molecular markers for qCTS4.
[0024] Figure 4 : Validation of the effectiveness of qCTS4 in seedling cold tolerance identification. Detailed Implementation
[0025] Example 1: Identification of qCTS4, a cold tolerance locus in rice seedlings, and its specific molecular marker.
[0026] I. Identification of the cold tolerance locus qCTS4 in rice seedlings
[0027] 1. Test materials and phenotypic identification
[0028] We used 372 rice germplasm accessions (163 indica and 209 japonica) provided by the Chinese Academy of Agricultural Sciences as materials. Disinfection, soaking, and germination were performed using conventional methods. Seedlings were cultured in a greenhouse or outdoor environment at 20-30℃. After the seedlings reached the 3-4 leaf stage, they were placed in an artificial climate chamber at 5℃ for 7 days. After the low-temperature treatment, the materials were moved to a greenhouse or outdoor environment with 20-30℃ light to recover. Leaf redness and wilt were assessed after 7 days, with the experiment repeated three times. Cold tolerance was evaluated based on the redness and wilt at 1, 3, 5, 7, and 9 leaves.
[0029] 2. Genome-wide association analysis of cold tolerance in rice seedlings
[0030] Genome-wide association analysis (GWAS) was performed using 3,566,872 SNPs from 372 rice whole genomes and seedling cold tolerance 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 negative result. -5 As a significance threshold, a locus qCTS4 was identified at 32446985 bp on chromosome 4 that was significantly associated with seedling cold tolerance. Figure 1 A and Figure 1 B).
[0031] 3. Allelic variation analysis at the qCTS4 locus
[0032] To examine the correlation between qCTS4 and seedling cold tolerance, this study compared the seedling cold tolerance of germplasm carrying different allelic variations at this locus. It was found that the seedling cold tolerance of germplasm carrying the A nucleotide allelic variation was significantly lower than that of germplasm carrying the G nucleotide allelic variation. Figure 2 (p < 0.001).
[0033] II. Development of qCTS4-specific molecular markers
[0034] 1. Design of KASP primers
[0035] Based on the nucleotide differences of qCTS4 at the physical location of 32,446,985 bp, the reference genome of ZS97 was extracted from the Ensembl genome database (http: / / ftp.ensemblgenomes.org / pub / release59 / plants / fasta / oryza_sativa_zs97 / ). The sequences within 100 bp above and below 32,446,985 bp were obtained using SnapGene software. Specific KASP markers for qCTS4 were designed using Primer3Plus (https: / / www.primer3plus.com / ). These included a seedling-specific primer qCTS4(A) for strong cold tolerance allelic variation, a seedling-specific primer qCTS4(G) for weak cold tolerance allelic variation, and a universal primer qCTS4(Common).
[0036] The primer sequence for the molecular marker qCTS4 (A) is as follows:
[0037] 5'-GAAGGTGACCAAGTTCATGCTCTATCAGGTGTAGGGCCCAGA-3' (SEQ ID No. 1),
[0038] qCTS4 (G) primer sequence:
[0039] 5'-GAAGGTCGGAGTCAACGGATTTATCAGGTGTAGGGCCCAGG-3' (SEQ ID No. 2),
[0040] qCTS4 (Common) primer sequence: 5'-AGGGGTGGAGCCAGGTGTGG-3' (SEQ ID No.3).
[0041] Example 2: Method for identifying cold tolerance in rice seedlings
[0042] 1. Methods for identifying the cold tolerance of rice seedlings.
[0043] 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.
[0044] 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.
[0045] Preparation of molecular marker primers: (1) The primers were purified by ULTRPAGE; (2) The primer powder was dissolved, and the concentrations of qCTS4(A) and qCTS4(G) were 36 μmol / μL, and the concentration of qCTS4(Common) was 90 μmol / μL; (3) The three primers were then mixed in a volume ratio of 1:1:1 to form KASP primer Mix.
[0046] 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.
[0047] 2. Identify the seedling cold tolerance of the tested rice varieties based on the nucleotide sequence of the PCR product:
[0048] If the PCR product is classified as type A, then the rice being tested is or is a candidate rice variety with strong cold tolerance in the seedling stage.
[0049] If the PCR product is classified as G, then the rice being tested is or is a candidate rice variety with weak cold tolerance during the seedling stage.
[0050] Example 3: Application of qCTS4-specific molecular marker in the identification of cold tolerance in rice seedlings
[0051] I. Test Materials
[0052] The test materials were: Gonggu, Hangzhou Nuo, Huangla Nuo, Gouhuanggang, Zhaoxing Hongmang Bainuo, Indica Nuo, Caoxiejin, Aiwudao, Xuxuwangu, Ganzigu, Baiyangdao, Gaogan Nuo, Baizhan Nuo, Niannuo, Baihujing, Hangu, Jixue Nuo, Zhakou Nuo, Baihe, Huanghugandan Nian, Wuyang Gaonuo, Honghandao, Huangnuo, Huanghu Indica Nuo, Haocu Nuo, Hongcaoguo Nuo, Hangu, Hehuakecaoguo Nuo, Hongke Nuo, and Houtunlian.
[0053] II. Identification of Cold Tolerance in Rice Seedlings
[0054] The seedling cold resistance of the test materials was determined according to the method in step 1 of Example 1.
[0055] The results are shown in Table 1. The table shows that among the 30 rice varieties, the average seedling cold tolerance level of Gonggu, Hangzhou Nuo, Huangla Nuo, Gouhuanggang, Zhaoxing Hongmang Bainuo, Xiandao Nuo, Caoxiejin, Aiwudao, Xuxu Wangu, Ganzigu, Baiyangdao, Gaogan Nuo, Baizhan Nuo, and Niannuo was 3.03, indicating strong seedling cold tolerance. The average seedling cold tolerance level of Baike Jing, Hangu, Jixuen Nuo, Zhakou Nuo, Baihe, Huangke Gandan Nian, Wumang Gaonuo, Honghandao, Huangnuo, Huangke Xiannuo, Haocu Nuo, Hongcaoguo Nuo, Hangu, Hehuake Caoguo Nuo, Hongke Nuo, and Houtunlian was 7.60, indicating weak seedling cold tolerance.
[0056] Table 1. Genotype and seedling cold tolerance identification results of the tested rice varieties
[0057]
[0058]
[0059] III. Genotyping
[0060] Genomic DNA was extracted from the test material in step one. Using the obtained genomic DNA as a template, genotyping was performed using the qCTS4-specific molecular marker. The specific experimental procedure is the same as step 1 in Example 2.
[0061] Allelic variations at the physical location 32446985 bp on chromosome 4 were detected using the qCTS4 specific molecular marker. The results are shown in Table 1 and [Table data would be inserted here]. Figure 3As shown, the PCR products of Gonggu, Hangzhou Nuo, Huangla Nuo, Gouhuanggang, Zhaoxing Hongmang Bainuo, Xiandao Nuo, Caoxiejin, Aiwudao, Xuxu Wangu, Ganzigu, Baiyangdao, Gaogan Nuo, Bai Nian Nuo, and Nian Nuo were all type A. According to the identification method for cold tolerance of rice seedlings in Example 2, Gonggu, Hangzhou Nuo, Huangla Nuo, Gouhuanggang, Zhaoxing Hongmang Bainuo, Xiandao Nuo, Caoxiejin, Aiwudao, Xuxu Wangu, Ganzigu, Baiyangdao, Gaogan Nuo, Bai Nian Nuo, and Nian Nuo are all rice varieties with strong cold tolerance at the seedling stage. Baike Jing, Hangu, and Ji... The PCR products of Xuenuo, Zhakounuo, Baihe, Huangkegandanzhan, Wuyanggaonuo, Honghandao, Huangnuo, Huangkexiannuo, Haocunuo, Hongcaoguonuo, Hangu, Hehuakecaoguonuo, Hongkenuo, and Houtunlian were all classified as G-type. According to the identification method for cold resistance of rice seedlings in Example 2, Baikejing, Hangu, Jixuenuo, Zhakounuo, Baihe, Huangkegandanzhan, Wuyanggaonuo, Honghandao, Huangnuo, Huangkexiannuo, Haocunuo, Hongcaoguonuo, Hangu, Hehuakecaoguonuo, Hongkenuo, and Houtunlian were all rice varieties with weak cold resistance in the seedling stage.
[0062] Therefore, it can be seen that the method for identifying the cold tolerance of rice seedlings in this invention is completely consistent with the results of the cold tolerance identification in step two, and the cold tolerance of rice varieties with allelic variation A (mean cold tolerance grade of 3.03) is significantly stronger than that of rice varieties with allelic variation G (mean cold tolerance grade of 7.60). Figure 4 (p < 0.001). This demonstrates that the method for identifying the cold tolerance of rice seedlings in this invention is accurate and reliable.
[0063] 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 molecular markers of cold tolerance in rice seedlings, characterized in that, The seedling cold tolerance of rice germplasm carrying A nucleotide allelic variants at the molecular marker sites for cold tolerance in the seedling stage was significantly stronger than that of germplasm carrying G nucleotide allelic variants. KASP primers were used for detection, and the KASP primers included specific primers for detecting seedling strong cold-resistant allelic variants carrying A nucleotide allelic variant sites, specific primers for detecting seedling weak cold-resistant allelic variants carrying G nucleotide allelic variants, and universal primers. The specific primer for detecting seedling-stage cold-resistant allelic variants carrying A nucleotide allelic variant sites is: 5'-GAAGGTGACCAAGTTCATGCTCTATCAGGTGTAGGGCCCAGA-3'. The specific primer used to detect seedling-stage weak cold-resistant allelic variants carrying G nucleotide allelic variations is: 5'-GAAGGTCGGAGTCAACGGATTTATCAGGTGTAGGGCCCAGG-3'. The universal primer is: 5'-AGGGGTGGAGCCAGGTGTGG-3'.
2. The detection method as described in claim 1, characterized in that: The detection method steps are as follows: extract genomic DNA from the rice to be tested, use the genomic DNA as a template, and perform PCR amplification using the KASP primers to obtain PCR products.
3. The detection method as described in claim 2, characterized in that: In the PCR amplification system, the concentrations of the seedling-stage cold-resistant allelic specific primers for detecting A nucleotide allelic variants and the seedling-stage weakly cold-resistant allelic variants for detecting G 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.
4. The application of the detection method according to any one of claims 1 to 3 in identifying the cold tolerance of rice seedlings, characterized in that, The molecular markers obtained using the aforementioned detection method were used to determine the cold tolerance of rice seedlings. Seedling cold tolerance was significantly stronger in germplasm carrying the A nucleotide allelic variant at the specified site than in germplasm carrying the G nucleotide allelic variant.
5. The application of the detection method according to any one of claims 1 to 3 in the cultivation of cold-resistant rice seedlings, characterized in that, The aforementioned detection method is used to assist in judging the cold tolerance of rice seedlings during the rice cultivation process. Rice varieties with strong cold tolerance during the seedling stage are selected, among which the seedling cold tolerance of germplasm carrying the A nucleotide allelic variant at this site is significantly stronger than that of germplasm carrying the G nucleotide allelic variant.