qST9, a locus of rice seed storage tolerance, and its molecular markers and applications

Abstract

This invention belongs to the field of agricultural biotechnology engineering, specifically disclosing the rice seed storage tolerance locus qST9, its molecular marker, and its application. The molecular marker is a site located at 11748764 bp on chromosome 9 that is significantly associated with storage tolerance. qST9 Furthermore, the storage tolerance of rice varieties carrying the G nucleotide allelic variant at this site is significantly higher than that of varieties carrying the A nucleotide allelic variant. This invention identifies the storage tolerance of rice varieties based on the nucleotide sequence of the PCR product: if the PCR product is type G, the rice variety is or a candidate variety with high storage tolerance; if the PCR product is type A, the rice variety is or a candidate variety with low storage tolerance. The method for identifying the storage tolerance of rice in this invention has been verified to be accurate and reliable.

Description

Technical Field

[0001] This invention belongs to agricultural biotechnology engineering, specifically relating to rice storage tolerance sites. qST9 And its molecular markers and applications. Background Technology

[0002] Food security is the foundation of the nation. During storage, rice seeds suffer significant reductions in vigor and quality due to factors such as storage conditions, environmental influences, and genetic factors. Therefore, improving seed storage tolerance and mitigating the decline in seed vigor and nutrient loss during storage are crucial for minimizing grain losses in my country and are of paramount strategic importance for ensuring national food security.

[0003] Seed storage tolerance is a complex quantitative trait influenced by both genetic and environmental factors. Therefore, conventional breeding methods based on phenotypic selection are insufficient for effectively selecting rice varieties with improved storage tolerance. With the development of quantitative genetics and molecular biology, the genetic basis regulating rice storage tolerance has been analyzed. Molecular design breeding, utilizing gene loci associated with rice storage tolerance and closely linked molecular markers, has enabled the efficient and accurate development of superior rice varieties with excellent storage tolerance. Summary of the Invention

[0004] This invention, through research, discovered a locus at 11,748,764 bp on chromosome 9 of rice that is significantly associated with storage tolerance. qST9 It was discovered that germplasm carrying G nucleotide allelic variations exhibited significantly higher storage tolerance than germplasm carrying A nucleotide allelic variations, thus leading to the completion of this invention.

[0005] This invention provides a molecular marker for rice storage tolerance, which is a site located at 11748764 bp on chromosome 9 that is significantly associated with storage tolerance. qST9 Specifically, the nucleotide difference is located at the physical position of 11,748,764 bp, and the storage tolerance of germplasm carrying the G nucleotide allelic variation at this site is significantly higher than that of germplasm carrying the A nucleotide allelic variation.

[0006] The present invention provides primers for detecting the aforementioned molecular markers, preferably KASP primers.

[0007] Specifically, this includes highly storable allelic variant-specific primers for detecting the G nucleotide allelic variant sites. qST9 -G, Specific primers for detecting low-storage-resistance allelic variants carrying A nucleotide allelic variants. qST9 -A, optional, also includes universal primers. qST9 -C.

[0008] More specifically, the qST9 -G primers are as follows:

[0009] 5'-GAAGGTCGGAGTCAACGGATTGTAGGAGTGACACGGAGCGG-3' (SEQ ID NO. 1),

[0010] qST9 -A primers are as follows:

[0011] 5'-GAAGGTGACCAAGTTCATGCTAGTAGGAGTGACACGGAGCGA-3' (SEQ ID NO. 2),

[0012] qST9 -C primers are as follows:

[0013] 5'-ACAGTGCTGGCCTCCCTCCG-3' (SEQ ID NO. 3).

[0014] This invention provides a method for detecting the molecular markers of rice storage tolerance, which uses the primers described above for detection, and the detection method is gene sequencing or molecular amplification.

[0015] The detection method described above is molecular amplification, and the steps of the method 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 primers to obtain PCR products.

[0016] Specifically, in the detection method described above, the primers in the PCR amplification system... qST9 -G and qST9 The concentration of -A is 30-45 μmol / μL. qST9 The -C concentration is 80-100 μmol / μL. The three primers are mixed in a volume ratio of 1:1:1 to form 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.

[0017] The amplification program was as follows: (1) pre-denaturation at 94℃ for 15 min; (2) denaturation at 94℃ for 20 s, extension at 61℃ for 60 s, and a decrease at a rate of 0.6℃ / cycle for 10 cycles; (3) denaturation at 94℃ for 20 s, extension at 55℃ for 60 s, and 26 cycles.

[0018] In addition, the present invention also provides the application of the primers or the detection method described herein in identifying rice plants or varieties with storage tolerance.

[0019] The application described herein includes the following steps for identifying rice plants or varieties with high storage tolerance: identifying the storage tolerance of the rice to be tested based on the nucleotide sequence of the PCR product: if the PCR product is classified as type G, then the rice to be tested is or is a candidate rice plant or variety with high storage tolerance; if the PCR product is classified as type A, then the rice to be tested is or is a candidate rice plant or variety with low storage tolerance.

[0020] This invention is based on the discovery of a locus at 11748764 bp on chromosome 9 of rice that is significantly associated with storage tolerance. qST9 The established method for identifying rice storage tolerance has been verified to be accurate and reliable. It can be used for efficient identification of rice variety storage tolerance or molecular design breeding of rice varieties with strong storage tolerance, and has application value. Attached Figure Description

[0021] Figure 1 The results of genome-wide association analysis (GWAS) of 234 rice accessions are shown. A and B represent the Manhattan plot and QQ plot of the germination potential index GWAS results, respectively, and C and D represent the Manhattan plot and QQ plot of the germination rate index GWAS results, respectively. qST9 Mark it with an arrow in the diagram.

[0022] Figure 2 : qST9 Comparison of germination rate index (A) and germination potential index (B) of storage-related traits between two allelic variant germplasms.

[0023] Figure 3 : qST9 The detection results of specific molecular markers.

[0024] Figure 4 qST9 Validation of the effectiveness of germination rate index (A) and germination potential index (B) in identifying storage-related traits. Detailed Implementation

[0025] Example 1: Rice storage tolerance sites qST9 Identification and its specific molecular markers

[0026] I. Sites for Rice Storage Tolerance qST9 Identification

[0027] 1. Test materials and phenotypic identification

[0028] Using 234 rice germplasm accessions provided by the Chinese Academy of Agricultural Sciences as materials, we treated the seeds at 42℃ and 75% humidity for 18 days. We then measured the germination rate and germination potential of the seeds under both treated and untreated conditions, and calculated the germination rate index (germination rate index = treatment germination rate / control germination rate * 100%) and the germination potential index (germination potential index = treatment germination potential / control germination potential * 100%).

[0029] 2. Genome-wide association analysis of rice storage tolerance

[0030] Genome-wide association analysis (GWAS) was performed using 2,541,213 SNPs from 234 rice genome accessions, along with germination rate and germination potential indices. 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. -5 As a significance threshold, a locus significantly associated with storage tolerance was identified at 11,748,764 bp on chromosome 9. qST9 ( Figure 1 ).

[0031] 3. qST9 Allelic variation analysis of loci

[0032] To test qST9 Correlation with storage tolerance was compared in this study with germplasm carrying different allelic variations at this locus. It was found that the germination potential index and germination rate index of germplasm carrying the G nucleotide allelic variation were significantly higher than those carrying the A nucleotide allelic variation. Figure 2 , p <0.001).

[0033] two, qST9 Development of specific molecular markers

[0034] 1. Design of KASP primers

[0035] according to qST9Nucleotide differences at physical location 11,748,764 bp were identified. A reference genome for 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 11,748,764 bp were obtained using SnapGene software. A targeting algorithm was designed using Primer3Plus (https: / / www.primer3plus.com / ). qST9 Specific KASP markers, including storage-resistant allelic variant-specific primers. qST9 (G) Specific primers for storage-intolerant allelic variations qST9 (A) and universal primers qST9 (Common).

[0036] The molecular marker qST9 (G) Primer sequence:

[0037] 5'-GAAGGTCGGAGTCAACGGATTGTAGGAGTGACACGGAGCGG-3' (SEQ ID NO. 1),

[0038] qST9 (A) Primer sequence:

[0039] 5'-GAAGGTGACCAAGTTCATGCTAGTAGGAGTGACACGGAGCGA-3' (SEQ ID NO. 2),

[0040] qST9 (Common) Primer sequence: 5'-ACAGTGCTGGCCTCCCTCCG-3' (SEQ ID NO.3).

[0041] Example 2: Method for identifying the storage tolerance of rice

[0042] 1. Methods for identifying the storage tolerance of rice varieties under test

[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. qST9 (G) and qST9 The concentration of (A) is 36 μmol / μL. qST9 (Common) concentration is 90 μmol / μL; (3) Then the three primers are mixed in a volume ratio of 1:1:1 to form KASP primer Mix.

[0046] The KASP genotyping experiment was performed as follows: A 96-well plate was used for the reaction and loading. 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 plate was 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, 10 cycles; (3) 94℃ denaturation for 20 s, 55℃ extension for 60 s, 26 cycles; (4) Data reading and analysis.

[0047] 2. Identify the storage tolerance of the rice species based on the nucleotide sequence of the PCR product:

[0048] If the PCR product is classified as G, then the rice being tested is or is a candidate rice variety with high storage tolerance.

[0049] If the PCR product is classified as type A, then the rice being tested is or is a candidate rice variety with low storage tolerance.

[0050] Example 3 qST9 Application of specific molecular markers in the identification of storage tolerance of rice varieties

[0051] I. Test Materials

[0052] The tested materials were CT 18657-2-1-2-1-2, Ganwanxian 23, Fengyuan Xiangdao, 52180, MR360, Huanghezhan, Menglai Xiangmi, R5, Donglian 5, Zhenyou 1, Shakewan 1, IR 78091-6-2-3-1-1, CT 18620-6-5-5-2-2, Shuangchao 25, Teqing, Dwarf Fengxin Red Rice, Ganwanxian 37, R9000, Jiaxing 8, HR-12, TG52, Huangruanzhan, Qixinzhan, CNR 39, CNR 49, Xiangwanxian 9, H17B 7, H17B 20, X17B 3, X17B 5, X17B 7, X17B 8, Daxiangnuo, and Radiation 632.

[0053] II. Identification of Rice Storage Tolerance

[0054] The storage resistance of the test material 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 34 rice varieties, CT 18657-2-1-2-1-2, Ganwanxian 23, Fengyuan Xiangdao, 52180, MR360, Huanghezhan, Menglai Xiangmi, R5, Donglian 5, Zhenyou 1, Shakewan 1, IR78091-6-2-3-1-1, and CT 18620-6-5-5-2-2 had an average germination potential index of 48.02% and an average germination rate index of 79.53%, indicating they are all rice varieties with high storage tolerance. Other varieties with high storage tolerance include Shuangchao 25, Teqing, Aihua Fengxin Hongmi, Ganwanxian 37, R9000, Jiaxing 8, HR-12, TG52, Huangruanzhan, Qixinzhan, CNR 39, CNR 49, Xiangwanxian 9, H17B 7, and H17B. The average germination potential index of 20, X17B3, X17B5, X17B7, X17B8, Da Xiang Nuo and Radiation 632 was 1.33%, and the average germination rate index was 4.30%, all of which are rice varieties with low storage tolerance.

[0056] Table 1. Genotyping and Storage Tolerance Identification Results of Tested Rice Varieties

[0057] ;

[0058]

[0059] III. Genotyping

[0060] Genomic DNA was extracted from the test material in step one, and the obtained genomic DNA was used as a template for... qST9 The specific molecular markers were used for typing. The specific experimental procedure is the same as step 1 in Example 2.

[0061] use qST9 Specific molecular markers were used to detect the allelic variation at physical location 11748764 bp on chromosome 9. The results are shown in Table 1 and... Figure 3 As shown, the PCR products of CT 18657-2-1-2-1-2, Ganwanxian 23, Fengyuan Xiangdao, 52180, MR360, Huanghezhan, Menglai Xiangmi, R5, Donglian 5, Zhenyou 1, Shakewan 1, IR 78091-6-2-3-1-1, and CT 18620-6-5-5-2-2 were all classified as G type. They were identified according to the method for identifying the storage tolerance of rice in Example 2. 18620-6-5-5-2-2 are all rice varieties with high storage tolerance. The PCR products of Shuangchao 25, Teqing, Dwarf Fengxin Red Rice, Ganwanxian 37, R9000, Jiaxing 8, HR-12, TG52, Huangruanzhan, Qixinzhan, CNR39, CNR49, Xiangwanxian 9, H17B7, H17B20, X17B3, X17B5, X17B7, X17B8, Daxiangnuo, and Radiation 632 were all type A. They were identified according to the rice storage tolerance identification method in Example 2. The PCR products of Shuangchao 25, Teqing, Dwarf Fengxin Red Rice, Ganwanxian 37, R9000, Jiaxing 8, HR-12, TG52, Huangruanzhan, Qixinzhan, and CNR49 were all type A. 39, CNR49, Xiangwanxian 9, H17B 7, H17B 20, X17B 3, X17B 5, X17B 7, X17B 8, Daxiangnuo, and Radiant 632 are all rice varieties with low storage tolerance.

[0062] Therefore, it can be seen that the method for identifying the storage tolerance of rice in this invention is completely consistent with the results of the storage tolerance identification in step two. Furthermore, the average germination potential index and average germination rate index of rice varieties with allelic variation G are 48.02% and 79.53% respectively, which are significantly higher than those of rice varieties with allelic variation A (average germination potential index 1.33%, average germination rate index 4.30%). Figure 4 , p <0.001). This demonstrates that the method for identifying the storage tolerance of rice 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 rice storage tolerance, characterized in that, The molecular marker for rice storage tolerance is a locus located at 11748764 bp on chromosome 9 that is significantly associated with storage tolerance. qST9 Specifically, the nucleotide difference is located at the physical position of 11748764 bp, and the storage tolerance of germplasm carrying the G nucleotide allelic variation at this site is significantly higher than that of germplasm carrying the A nucleotide allelic variation. KASP primers were used for detection.

2. The detection method as described in claim 1, characterized in that, The KASP primers include specific primers for detecting the highly storage-resistant allelic variants carrying the G nucleotide allelic variant sites. qST9 -G, Specific primers for detecting low-storage-resistance allelic variants carrying A nucleotide allelic variants. qST9 -A and universal primers qST9 -C; where, Specific primers qST9 -G is: 5'-GAAGGTCGGAGTCAACGGATTGTAGGAGTGACACGGAGCGG-3', Specific primers qST9 -A is: 5'-GAAGGTGACCAAGTTCATGCTAGTAGGAGTGACACGGAGCGA-3', universal primers qST9 -C is: 5'-ACAGTGCTGGCCTCCCTCCG-3'.

3. The detection method as described in claim 1 or 2, characterized in that, The detection method is gene sequencing or molecular amplification.

4. The detection method as described in claim 3, characterized in that, 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, and perform PCR amplification using the KASP primers to obtain PCR products.

5. The detection method as described in claim 4, characterized in that, In the PCR amplification system, the primers qST9 -G and qST9 The concentration of -A is 30-45 μmol / μL. qST9 The -C concentration is 80-100 μmol / μL. The three primers are mixed in a volume ratio of 1:1:1 to form KASP primer Mix. The total reaction volume is 10.14 μL, and the reaction system includes: 5 μL DNA, 5 μL 2x KASPMaster Mix, and 0.14 μL KASP primer Mix. The amplification program was as follows: (1) pre-denaturation at 94℃ for 15 min; (2) denaturation at 94℃ for 20 s, extension at 61℃ for 60 s, and a decrease at a rate of 0.6℃ / cycle for 10 cycles; (3) denaturation at 94℃ for 20 s, extension at 55℃ for 60 s, and 26 cycles.

6. The application of the detection method as described in any one of claims 1-5 in identifying rice plants or varieties with storage tolerance.

7. The application as described in claim 6, characterized in that, The steps for identifying rice plants or varieties with high storage tolerance include: identifying the storage tolerance of the rice to be tested based on the nucleotide sequence of the PCR product: if the PCR product is classified as G, the rice to be tested is or is a candidate rice plant or variety with high storage tolerance; if the PCR product is classified as A, the rice to be tested is or is a candidate rice plant or variety with low storage tolerance.

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