Rice seed storability locus qST9 as well as molecular marker and application thereof

Abstract

The invention belongs to agricultural biotechnology engineering, and particularly discloses a rice seed storability locus qST9 as well as a molecular marker and application thereof. The molecular marker is a locus qST9 which is positioned at 11748764 bp of a No.9 chromosome and is remarkably related to storage resistance, and the storage resistance of a germplasm carrying G nucleotide allelic variation at the locus is remarkably higher than that of a germplasm carrying A nucleotide allelic variation. According to the invention, the storage tolerance of rice to be detected is identified according to the nucleotide sequence of a PCR product; if the typing of the PCR product is G type, the rice to be detected is a rice plant or a rice variety with higher storage tolerance or a candidate rice plant or a candidate rice variety with higher storage tolerance; if the typing of the PCR product is type A, the to-be-detected rice is a rice plant or variety with relatively low storage tolerance or a candidate rice plant or variety with relatively low storage tolerance. The method for identifying the storability of the rice is verified to be accurate and reliable.

Description

Technical Field

[0001] The present invention belongs to agricultural biotechnology engineering, and specifically relates to rice storage tolerance sites qST9 Its molecular markers and applications. Background Art

[0002] Grain security is the foundation of the nation. Rice seed storage can severely reduce seed vigor and grain quality due to factors such as storage conditions, environmental factors, and genetics. Therefore, improving seed storage tolerance and mitigating the decline in vigor and nutrient loss during storage, thereby reducing stored grain losses, is of strategic importance for ensuring national food security.

[0003] Seed storability is a complex quantitative trait influenced by both genetic and environmental factors. Consequently, conventional breeding based on phenotypic selection has been unable to effectively select rice varieties with storability. Advances in quantitative genetics and molecular biology have enabled the analysis of the genetic basis regulating rice storability. By utilizing genetic loci associated with rice storability and closely linked molecular markers, molecular design breeding has enabled the efficient and accurate development of superior storability rice varieties. Summary of the Invention

[0004] The present invention has found that there is a locus significantly related to storage tolerance at 11748764 bp on chromosome 9 of rice. qST9 , found that the storage tolerance of germplasm carrying G nucleotide allele variation was significantly higher than that of germplasm carrying A nucleotide allele variation, and thus completed the present invention.

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

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

[0007] Specifically, it includes a high storage resistance allele variant specific primer for detecting the G nucleotide allele variant site. qST9 -G, primers specific for low storage stability alleles carrying A nucleotide alleles qST9 -A, optionally also includes a universal primer qST9 -C.

[0008] More specifically, the qST9 -G primers are as follows: 5'-GAAGGTCGGAGTCAACGGATTGTAGGAGTGACACGGAGCGG-3' (SEQ ID NO. 1), qST9 -A primers are as follows: 5'-GAAGGTGACCAAGTTCATGCTAGTAGGAGTGACACGGAGCGA-3' (SEQ ID NO. 2), qST9 -C primers are as follows: 5'-ACAGTGCTGGCCTCCCTCCG-3' (SEQ ID NO. 3).

[0009] The present invention provides a method for detecting the rice storage tolerance molecular marker, which adopts the primers for detection, and the detection method is gene sequencing or molecular amplification.

[0010] The above detection method is molecular amplification, and the steps of the method are as follows: extracting genomic DNA from the rice to be tested, using the genomic DNA as a template and the primers to perform PCR amplification to obtain a PCR product.

[0011] The detection method, specifically, in the PCR amplification system, the primers qST9 -G and qST9 -A concentration is 30-45 μmol / μL, qST9 The concentration of -C was 80-100 μmol / μL, and the three primers were mixed at a volume ratio of 1:1:1 to form KASP Primer Mix. The total reaction volume of the reaction system was 10.14 μL, and the reaction system included: 5 μL DNA, 5 μL 2x KASP Master Mix, and 0.14 μL KASP Primer Mix. The amplification program was as follows: (1) pre-denaturation at 94°C for 15 min; (2) denaturation at 94°C for 20 s, extension at 61°C for 60 s, ramping at 0.6°C / cycle, for 10 cycles; (3) denaturation at 94°C for 20 s, extension at 55°C for 60 s, for 26 cycles.

[0012] At the same time, the present invention also provides the application of the primers or the detection method in identifying rice plants or varieties with storage tolerance.

[0013] The application includes the step of identifying rice plants or varieties with good storage tolerance, comprising: identifying the storage tolerance of the rice to be tested based on the nucleotide sequence of the PCR product: if the PCR product is type G, the rice to be tested is or is a candidate for being a rice plant or variety with high storage tolerance; if the PCR product is type A, the rice to be tested is or is a candidate for being a rice plant or variety with low storage tolerance.

[0014] The present invention is based on the discovery that there is a locus significantly associated with storage tolerance at 11748764 bp on chromosome 9 of rice. qST9 The established method for identifying rice storage tolerance has been proven to be accurate and reliable. It can be used for the efficient identification of rice varieties' storage tolerance or the molecular design breeding of rice varieties with strong storage tolerance, and has application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : 234 rice genome-wide association analysis (GWAS) results, wherein A and B represent the Manhattan plot and QQ plot of the germination index GWAS results, respectively, C and D represent the Manhattan plot and QQ plot of the germination rate index GWAS results, respectively, qST9 Marked with arrows in the figure.

[0016] Figure 2 : qST9 Comparison of germination rate index (A) and germination potential index (B) of storage-related traits between two allelic variant germplasms.

[0017] Figure 3 : qST9 The detection results of specific molecular markers.

[0018] Figure 4 :qST9 Verification of the effectiveness in the identification of germination rate index (A) and germination potential index (B) for storage-related traits. DETAILED DESCRIPTION

[0019] Example 1: Rice storage tolerance locus qST9 Identification and specific molecular markers 1. Rice Storage Tolerance Loci qST9 Identification 1. Test Materials and Phenotypic Identification We used 234 rice germplasms provided by the Chinese Academy of Agricultural Sciences as materials. After treating the seeds at 42°C and 75% humidity for 18 days, we measured the germination rate and germination potential of the seeds under treated and untreated conditions, and calculated the germination rate index (germination rate index = treatment germination rate / control germination rate*100%) and germination potential index (germination potential index = treatment germination potential / control germination potential*100%).

[0020] 2. Genome-wide association analysis of rice storage tolerance A genome-wide association study (GWAS) was performed using 2,541,213 SNP data from 234 rice genomes and data on germination rate and germination potential. The GWAS was performed 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.). The kinship matrix and population structure Q matrix of the population were combined to calculate the mixed linear model (MLM). P < 10 -5 As the significance threshold, a locus significantly associated with storage tolerance was identified at 11748764 bp on chromosome 9. qST9 ( Figure 1 ).

[0021] 3. qST9 Allelic variation analysis of the locus To test qST9 Correlation between storage resistance and storability. This study compared the storage resistance of germplasms carrying different alleles at this site. It was found that the germination index and germination rate index of storage resistance-related traits of germplasms carrying G nucleotide allele were significantly higher than those carrying A nucleotide allele ( Figure 2 , p <0.001).

[0022] two, qST9 Development of specific molecular markers 1. Design of KASP primers according to qST9 The nucleotide difference at the physical position 11748764 bp was analyzed. 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 of 100 bp above and below 11748764 bp were obtained using SnapGene software. Primer3Plus (https: / / www.primer3plus.com / ) was used to design the primers for the ZS97 gene. qST9 Specific KASP markers for storage stability alleles. qST9 (G) Specific primers for storage-intolerant alleles qST9 (A) and universal primers qST9 (Common).

[0023] The molecular marker qST9 (G) Primer sequence: 5'-GAAGGTCGGAGTCAACGGATTGTAGGAGTGACACGGAGCGG-3' (SEQ ID NO. 1), qST9 (A) Primer sequences: 5'-GAAGGTGACCAAGTTCATGCTAGTAGGAGTGACACGGAGCGA-3' (SEQ ID NO. 2), qST9 (Common) primer sequence: 5′-ACAGTGCTGGCCTCCCTCCG-3′ (SEQ ID NO. 3).

[0024] Example 2: Method for Identifying Rice Storage Tolerance 1. Method for identifying storage tolerance of rice to be tested The details are as follows: extract genomic DNA from the rice to be tested, use the genomic DNA as a template, perform PCR amplification using molecular markers, and obtain PCR products; In the above experiment, the DNA extraction method steps are as follows: (1) Place a small amount of fresh rice leaves in a 2 mL centrifuge tube filled with steel balls, immerse it in liquid nitrogen for 10 minutes, and then quickly place it in a sampler and grind it into powder; (2) Add 600 μL of CTAB buffer and place it in a 65°C water bath for 30 minutes; (3) Add an equal volume of chloroform:isoamyl alcohol (24:1) solution and shake it vigorously to mix thoroughly; (4) Centrifuge at 12,000 rpm for 10 minutes, and remove 400 μL of the supernatant into a new 1.5 mL centrifuge tube; (5) Add 400 μL of pre-cooled isopropanol and place it in a -20°C refrigerator for at least 20 minutes to precipitate DNA; (6) Centrifuge at 12,000 rpm for 10 minutes, and discard the supernatant; (7) Air-dry at room temperature and add 400 μL of double-distilled sterile water (ddH2O) to dissolve the DNA.

[0025] Preparation of molecular marker primers: (1) Purify the primers by ULTRPAGE; (2) Dissolve the primer powder. 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.

[0026] The KASP genotyping experiment was performed as follows: The reaction was performed using a 96-well plate. The total reaction volume per 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 membrane that was centrifuged and then fluorescently examined. After confirming that there were no air holes, PCR amplification was performed. The amplification procedure was as follows: (1) 94°C pre-denaturation for 15 min; (2) 94°C denaturation for 20 s, 61°C extension for 60 s, and ramp rate of 0.6°C / cycle for 10 cycles; (3) 94°C denaturation for 20 s, 55°C extension for 60 s, and 26 cycles; (4) data reading and analysis.

[0027] 2. Identify the storage tolerance of the rice to be tested based on the nucleotide sequence of the PCR product: If the PCR product is type G, the rice to be tested is or is a candidate for a rice variety with higher storage tolerance; If the typing of the PCR product is type A, the rice to be tested is or is a candidate for a rice variety with low storage tolerance.

[0028] Example 3 qST9 Application of specific molecular markers in identification of storage tolerance of rice varieties 1. Test Materials The test materials were CT 18657-2-1-2-1-2, Ganwanxian 23, Fengyuanxiangdao, 52180, MR360, Huanghezhan, Menglaixiangmi, 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 Fengxinhongmi, 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 Radiated 632.

[0029] 2. Identification of Rice Storage Tolerance The storage stability of the test materials was identified according to the method in step 1 of Example 1.

[0030] The results are shown in Table 1. As can be seen from the table, among the 34 rice varieties, the average germination index of CT 18657-2-1-2-1-2, Ganwanxian 23, Fengyuanxiangdao, 52180, MR360, Huanghezhan, Menglaixiangmi, R5, Donglian 5, Zhenyou 1, Shakewan 1, IR78091-6-2-3-1-1, and CT 18620-6-5-5-2-2 was 48.02%, and the average germination rate index was 79.53%, all of which are rice varieties with high storage resistance; Shuangchao 25, Teqing, Dwarf Fengxinhongmi, 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, Daxiangnuo and Yuyi 632 was 1.33%, and the average germination rate index was 4.30%, indicating that all of them were rice varieties with low storage tolerance.

[0031] Table 1. Genotypes and storage tolerance identification results of the tested rice varieties ;

[0032] 3. Genotype identification Extract the genomic DNA of the test material in step 1, and use the obtained genomic DNA as a template. qST9 The specific experimental process is the same as step 1 of Example 2.

[0033] use qST9 Specific molecular markers were used to detect the allelic variation at the physical position 11748764 bp on chromosome 9. The test results are shown in Table 1 and Figure 3As shown, the PCR products of CT 18657-2-1-2-1-2, Ganwan Xianxian 23, Fengyuan Xiangdao, 52180, MR360, Huang Hezhan, 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 G-type. According to the identification method of rice storage resistance in Example 2, CT 18657-2-1-2-1-2, Ganwan Xianxian 23, Fengyuan Xiangdao, 52180, MR360, Huang Hezhan, 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 identified as G-type. 18620-6-5-5-2-2 are all rice varieties with high storage tolerance; Shuangchao 25, Teqing, dwarf Fengxin red rice, Ganwanxian 37, R9000, Jiaxing 8, HR-12, TG52, Huangruanzhan, Qixinzhan, CNR39, CNR 49, Xiangwanxian 9, H17B 7, H17B 20, X17B 3, X17B 5, X17B 7, X17B 8, Daxiangnuo and Irradiation 632 were all type A. According to the identification method for rice storage tolerance in Example 2, Shuangchao 25, Teqing, dwarf Fengxin red rice, Ganwanxian 37, R9000, Jiaxing 8, HR-12, TG52, Huangruanzhan, Qixinzhan, CNR 39, CNR49, Xiangwanxian 9, H17B 7, H17B 20, X17B 3, X17B 5, X17B 7, X17B 8, Daxiangnuo and Yiwu 632 are all rice varieties with low storage tolerance.

[0034] It can be seen from this that the rice storage resistance identification method of the present invention is completely consistent with the storage resistance identification results in step 2, and the storage resistance-related traits of rice varieties with allelic variation G have an average germination potential index of 48.02% and an average germination rate index of 79.53%, which are significantly higher than the storage resistance of rice varieties with allelic variation A (the average germination potential index is 1.33% and the average germination rate index is 4.30%) ( Figure 4 , p <0.001). This indicates that the method for identifying the storage stability of rice of the present invention is accurate and reliable.

[0035] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A molecular marker for rice storage tolerance, characterized in that: It is a locus located at 11748764 bp on chromosome 9 that is significantly associated with storage stability. qST9 Specifically, the nucleotide difference is at the physical position 11748764 bp, and the storage tolerance of the germplasm carrying the G nucleotide allele at this site is significantly higher than that of the germplasm carrying the A nucleotide allele.

2. A primer for detecting the molecular marker according to claim 1, which is a KASP primer.

3. The primer according to claim 2, wherein It includes a high storage tolerance allele variation specific primer for detecting the G nucleotide allele variation site, a low storage tolerance allele variation specific primer for detecting the A nucleotide allele variation site, and optionally also includes a universal primer.

4. The primer according to claim 3, wherein The highly storage-resistant allele variant-specific primer for detecting the G nucleotide allele variant site qST9 -G means: 5'-GAAGGTCGGAGTCAACGGATTGTAGGAGTGACACGGAGCGG-3', Primers specific for low storage stability alleles for detecting alleles carrying A nucleotides qST9 -A is: 5'-GAAGGTGACCAAGTTCATGCTAGTAGGAGTGACACGGAGCGA-3', The universal primer qST9 -C is: 5'-ACAGTGCTGGCCTCCCTCCG-3'.

5. A method for detecting molecular markers of rice storage tolerance according to claim 1, characterized in that: The method is detected by using the primers according to any one of claims 2 to 4.

6. The detection method according to claim 5, wherein The detection method is gene sequencing or molecular amplification.

7. The detection method according to claim 6, wherein The detection method is molecular amplification, and the steps of the method are as follows: extracting genomic DNA of the rice to be tested, using the genomic DNA as a template, and performing PCR amplification using the primers according to any one of claims 2 to 4 to obtain a PCR product.

8. The detection method according to claim 7, wherein In the PCR amplification system, the primers qST9 -G and qST9 -A concentration is 30-45 μmol / μL, qST9 The concentration of -C was 80-100 μmol / μL, and the three primers were mixed at a volume ratio of 1:1:1 to form KASP Primer Mix. The total reaction volume of the reaction system was 10.14 μL, and the reaction system included: 5 μL DNA, 5 μL 2x KASP Master Mix, and 0.14 μL KASP Primer Mix. The amplification program was as follows: (1) pre-denaturation at 94°C for 15 min; (2) denaturation at 94°C for 20 s, extension at 61°C for 60 s, ramping at 0.6°C / cycle, for 10 cycles; (3) denaturation at 94°C for 20 s, extension at 55°C for 60 s, for 26 cycles.

9. Use of the primer according to any one of claims 2 to 4, or the detection method according to any one of claims 5 to 8, in identifying rice plants or varieties with storage tolerance.

10. The use according to claim 9, characterized in that The step of identifying rice plants or varieties with good storage resistance includes: identifying the storage resistance of the rice to be tested based on the nucleotide sequence of the PCR product: if the typing of the PCR product is G type, the rice to be tested is or is a candidate for a rice plant or variety with higher storage resistance; if the typing of the PCR product is A type, the rice to be tested is or is a candidate for a rice plant or variety with lower storage resistance.

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