Rice salt tolerance site qSST8 and its molecular markers and applications

Abstract

This invention belongs to the field of agricultural biotechnology engineering and discloses the rice salt tolerance locus qSST8, its molecular marker, and its application. The molecular marker is qSST8, a locus located at 6577904 bp on chromosome 8 that is significantly associated with salt tolerance. Furthermore, germplasm carrying the A nucleotide allelic variant at this locus exhibits significantly stronger salt tolerance than germplasm carrying the G nucleotide allelic variant. The method for identifying rice salt tolerance in this invention has been verified to be accurate and reliable.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology engineering, specifically relating to the rice salt tolerance site qSST8 and its molecular markers and applications. Background Technology

[0002] Global climate change, seawater intrusion, and improper irrigation have exacerbated soil salinization, leading to a decline in the productivity of many arable lands. Rice cultivation on saline-alkali land is a traditional method for improving saline-alkali soil, serving both as soil utilization and improvement, with the soil becoming increasingly suitable for rice cultivation. Therefore, improving the salt tolerance of rice and cultivating varieties adapted to saline-alkali environments can enable these traditionally unusable "barren lands" to generate food production, alleviate the shortage of arable land resources, and enhance the stability and resilience of food production.

[0003] Salt tolerance in rice is a complex quantitative trait regulated by multiple genes, making it difficult to accurately identify in the field. Conventional breeding methods struggle to efficiently and precisely screen for salt-tolerant varieties. This study aims to analyze key gene loci regulating rice salt tolerance and develop functional markers. Using molecular markers for precise selection allows for the efficient elimination of individuals lacking target genes in early generations, reducing the size of subsequent experimental populations, optimizing breeding design, improving selection efficiency, shortening the breeding cycle, enhancing selection accuracy and controllability, and driving the transformation of salt-tolerant breeding towards precision and efficiency. Summary of the Invention

[0004] Through research, this invention discovered a site qSST8 at 6577904 bp on chromosome 8 of rice that is significantly associated with salt tolerance. It was found that the salt tolerance of germplasm carrying the A nucleotide allelic variant is significantly stronger than that of germplasm carrying the G nucleotide allelic variant, thus completing this invention.

[0005] This invention provides a molecular marker for rice salt tolerance, which is the qSST8 site located at 6577904 bp on chromosome 8, which is significantly associated with salt tolerance. Specifically, it is the nucleotide difference at the physical location of 6577904 bp. Furthermore, the salt 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.

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

[0007] Specifically, the primers include salt-tolerant allelic variant-specific primers for detecting A nucleotide allelic variant sites, weakly salt-tolerant allelic variant-specific primers for detecting G nucleotide allelic variant sites, and universal primers.

[0008] More specifically, the salt-tolerant allelic specific primer for detecting the A nucleotide allelic variant site is as follows: 5'-GAAGGTCGGAGTCAACGGATTGATGAACTCCTTCGGCACTCCA-3' (SEQ ID No. 1).

[0009] The specific primers for detecting weakly salt-tolerant allelic variants carrying G nucleotide allelic variants are as follows: 5'-GAAGGTGACCAAGTTCATGCTATGAACTCCTTCGGCACTCCG-3' (SEQ ID No. 2).

[0010] The universal primer is as follows: 5'-CAGAAGTTCGTGTGGAAAAACATTGTTTG-3' (SEQ ID No. 3).

[0011] The present invention also provides a method for detecting the molecular marker of rice salt tolerance, which uses the primers described above for detection, and the detection method is gene sequencing or molecular amplification.

[0012] 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 primers to obtain PCR products.

[0013] The present invention also provides a method for identifying rice salt tolerance, which uses the method for detecting rice salt tolerance molecular markers to obtain the results of the molecular markers to determine the salt tolerance of rice, wherein the salt tolerance of the rice is significantly stronger than that of the rice carrying A nucleotide allelic variants at the site than that of the rice carrying G nucleotide allelic variants.

[0014] Specifically, the method is 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.

[0015] More specifically, in the PCR amplification system, the concentrations of qSST8-A and qSST8-G are 30-45 μmol / μL, the concentration of qSST8-C is 80-100 μmol / μL, and the three primers are mixed in a volume ratio of 1:1:1 to form the KASP primer mix; the reaction system has a total reaction volume of 10.14 μL and includes: 5 μL of DNA, 5 μL of 2x KASP Master Mix, and 0.14 μL of KASP primer mix;

[0016] 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.

[0017] The method further identifies the salt tolerance of the rice to be tested based on the nucleotide sequence of the PCR product: if the PCR product is type A, the rice to be tested is or is a candidate rice variety with strong salt tolerance; if the PCR product is type G, the rice to be tested is or is a candidate rice variety with weak salt tolerance.

[0018] The present invention also provides the application of the detection method described above in the cultivation of salt-tolerant rice. The detection method described above is used to assist in judging the salt tolerance of rice during the rice cultivation process, and to select rice with strong salt tolerance. Among them, the salt tolerance of the germplasm carrying the A nucleotide allelic variation at this site is significantly stronger than that of the germplasm carrying the G nucleotide allelic variation.

[0019] The method for identifying rice salt tolerance of the present invention has been verified to have high accuracy, good repeatability, simple operation, standardized process, short identification cycle, and high detection efficiency, and has wide application value. Attached Figure Description

[0020] Figure 1 The results of genome-wide association analysis (GWAS) of salt tolerance levels in 372 rice accessions are shown in the figure. A and B represent the Manhattan plot and QQ plot of the salt tolerance level GWAS results, respectively. qSST8 in this invention is marked with an arrow in the figure.

[0021] Figure 2 Comparison of salt tolerance levels between two allelic variants of qSST8.

[0022] Figure 3 : genotyping results of qSST8 specific molecular markers.

[0023] Figure 4 : Validation of the effectiveness of qSST8 in salt tolerance identification. Detailed Implementation

[0024] Example 1: Identification of the rice salt tolerance site qSST8 and its specific molecular marker

[0025] I. Identification of the rice salt tolerance locus qSST8

[0026] 1. Test materials and phenotypic identification

[0027] We used 372 rice germplasm accessions provided by the Chinese Academy of Agricultural Sciences as materials to conduct salt tolerance identification at the Binhai Agriculture Research Institute of the Hebei Academy of Agricultural and Forestry Sciences. We used the moist seedling raising method, transplanting at the three-leaf-one-heart stage, followed by continuous irrigation with saline water (conductivity ≥10 ms / cm) at a concentration of 0.5% NaCl. Salt tolerance was evaluated based on the redness and wilt of the 1st, 3rd, 5th, 7th, and 9th leaves. Surveys were conducted 2 weeks and 4 weeks after salt stress treatment. For each material, 10 consecutive plants were selected for individual evaluation, and the average salt tolerance level was calculated.

[0028] 2. Genome-wide association analysis of salt tolerance in rice

[0029] Genome-wide association analysis (GWAS) was performed using 3,566,872 SNPs and salt tolerance data from 372 rice whole genomes. 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. -4 As a significance threshold, a site qSST8 significantly associated with salt tolerance was identified at 6577904 bp on chromosome 8. Figure 1 (A and 1B).

[0030] 3. Allelic variation analysis at the qSST8 locus.

[0031] To examine the correlation between qSST8 and salt tolerance, this study compared the salt tolerance of germplasm carrying different allelic variations at this locus. It was found that the salt tolerance level 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), meaning that the salt tolerance of germplasm carrying A nucleotide allelic variation is significantly stronger than that of germplasm carrying G nucleotide allelic variation.

[0032] II. Development of qSST8-specific molecular markers

[0033] 1. Design of KASP primers

[0034] Based on the nucleotide differences of qSST8 at physical position 6577904 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 sequence within 100 bp above and below 6577904 bp was obtained using SnapGene software, and specific KASP markers for qSST8 were designed using Primer3Plus (https: / / www.primer3plus.com / ). These included a salt-tolerant allelic variant-specific primer qSST8(A), a weakly salt-tolerant allelic variant-specific primer qSST8(G), and a universal primer qSST8(Common).

[0035] The primer sequence for the molecular marker qSST8 (A) is as follows:

[0036] 5'-GAAGGTCGGAGTCAACGGATTGATGAACTCCTTCGGCACTCCA-3' (SEQ ID No. 1),

[0037] qSST8 (G) primer sequence:

[0038] 5'-GAAGGTGACCAAGTTCATGCTATGAACTCCTTCGGCACTCCG-3' (SEQ ID No. 2),

[0039] qSST8 (Common) primer sequence: 5'-CAGAAGTTCGTGTGGAAAAACATTGTTTG-3' (SEQ ID No. 3).

[0040] Example 2: Method for identifying salt tolerance in rice

[0041] 1. Methods for identifying the salt tolerance of rice varieties under test

[0042] 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.

[0043] 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.

[0044] Preparation of molecular marker primers: (1) The primers were purified by ULTRPAGE; (2) The primer powder was dissolved, and the concentrations of qSST8(A) and qSST8(G) were 36 μmol / μL, and the concentration of qSST8(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.

[0045] 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.

[0046] 2. Identify the salt tolerance of the rice variety based on the nucleotide sequence of the PCR product:

[0047] If the PCR product is classified as type A, then the rice being tested is or is a candidate rice variety with strong salt tolerance.

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

[0049] Example 3: Application of qSST8 specific molecular marker in the identification of salt tolerance in rice varieties

[0050] I. Test Materials

[0051] The test materials were sticky glutinous rice, dwarf glutinous rice, red-haired fragrant rice 1, bian rice, white sand glutinous rice, yellow-shelled glutinous rice, black rust glutinous rice, thousand-pound glutinous rice, cow-foot flat glutinous rice, black-shelled rice, dwarf black rice, red-shelled glutinous rice, dwarf glutinous rice, yellow-shelled glutinous rice, white fruit glutinous rice (1), tall stalk glutinous rice, October green, dry rice, white mustard grass, glutinous rice, Rongtang rice, Zhanli glutinous rice 1, Houtai, black glutinous rice, Jizi rice, flower glutinous rice, big fragrant glutinous rice, dry hairy rice, red-shelled late rice, and bald glutinous rice.

[0052] II. Identification of Salt Tolerance in Rice

[0053] The salt resistance of the test material was determined according to the method in step 1 of Example 1.

[0054] The results are shown in Table 1. As can be seen from the table, among the 30 rice varieties, the average salt tolerance level of glutinous rice, dwarf glutinous rice, red hairy fragrant rice 1, bian rice, white sand glutinous rice, yellow husk glutinous rice, black rust glutinous rice, thousand jin glutinous rice, ox foot flat glutinous rice, black husk rice, dwarf black rice, red husk glutinous rice, dwarf glutinous rice, yellow husk glutinous rice, and white fruit glutinous rice (1) is 3.43, which are all rice varieties with strong salt tolerance; the average salt tolerance level of tall glutinous rice, October green, dry rice, white mustard grass, glutinous rice, Rongtang rice, Zhanli glutinous rice 1, Houtai, black glutinous rice, Jizi rice, flower glutinous rice, big fragrant glutinous rice, dry hairy rice, red husk late rice, and bald head glutinous rice is 7.30, which are all rice varieties with weak salt tolerance.

[0055] Table 1. Genotyping and salt tolerance identification results of the tested rice varieties

[0056]

[0057]

[0058] III. Genotyping

[0059] Genomic DNA was extracted from the test material in step one. Using the obtained genomic DNA as a template, genotyping was performed using the qSST8 specific molecular marker. The specific experimental procedure is the same as step 1 in Example 2.

[0060] Allelic variations at the physical location 6577904 bp on chromosome 8 were detected using the qSST8 specific molecular marker. The results are shown in Table 1 and [Table data would be inserted here]. Figure 3As shown, the PCR products of sticky glutinous rice, dwarf glutinous rice, red-haired fragrant rice 1, brittle rice, white sand glutinous rice, yellow-shelled glutinous rice, black-rusted glutinous rice, thousand-pound glutinous rice, cow-foot flat glutinous rice, black-shelled rice, dwarf black rice, red-shelled glutinous rice, dwarf glutinous rice, yellow-shelled glutinous rice, and white glutinous rice (1) were all type A. They were identified according to the method for identifying rice salt tolerance in Example 2. Sticky glutinous rice, dwarf glutinous rice, red-haired fragrant rice 1, brittle rice, white sand glutinous rice, yellow-shelled glutinous rice, black-rusted glutinous rice, thousand-pound glutinous rice, cow-foot flat glutinous rice, black-shelled rice, dwarf black rice, red-shelled glutinous rice, dwarf glutinous rice, yellow-shelled glutinous rice, and white glutinous rice (1) all showed strong salt tolerance. The PCR products of the following rice varieties were all classified as G-type: Gaogan Nuo, Shiyueqing, Hanfangu, Baijiecao, Nuohe, Rongtanghe, Zhanli Nuohe 1, Houtai, Heinuogu, Jizigu, Huanuo, Daxiangnuo, Hanmaodao, Hongke Wangu, and Guangtou Nuo. The salt tolerance of rice was determined according to the method described in Example 2. All of these varieties were found to have relatively weak salt tolerance.

[0061] Therefore, it can be seen that the method for identifying rice salt tolerance in this invention is completely consistent with the salt tolerance identification results in step two, and the salt tolerance of rice varieties with allelic variation A (mean salt tolerance grade 3.43) is significantly stronger than that of rice varieties with allelic variation G (mean salt tolerance grade 7.30). Figure 4 (p < 0.001). This demonstrates that the method for identifying rice salt tolerance in this invention is accurate and reliable.

[0062] 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 identifying the salt tolerance of rice, characterized in that, Rice salt tolerance was determined by detecting molecular markers of salt tolerance. Germplasm carrying A nucleotide allelic variations at the specified salt tolerance molecular marker sites showed significantly stronger salt tolerance than germplasm carrying G nucleotide allelic variations. KASP primers were used to detect these markers. The KASP primers included specific primers for detecting strong salt tolerance allelic variations carrying A nucleotide allelic variations, specific primers for detecting weak salt tolerance allelic variations carrying G nucleotide allelic variations, and universal primers. The specific primer for detecting salt-tolerant allelic variants carrying nucleotide A allelic variant sites is: 5'-GAAGGTCGGAGTCAACGGATTGATGAACTCCTTCGGCACTCCA-3', The specific primers for detecting weakly salt-tolerant allelic variants carrying G nucleotide allelic variants are: 5'-GAAGGTGACCAAGTTCATGCTATGAACTCCTTCGGCACTCCG-3', The universal primer is: 5'-CAGAAGTTCGTGTGGAAAAACATTGTTTG-3'.

2. The method as described in claim 1, characterized in that, Genomic DNA was extracted from the rice to be tested. Using the genomic DNA as a template, PCR amplification was performed using the KASP primers to obtain the PCR product. Salt tolerance of rice can be determined by the nucleotide sequence of the PCR product: if the PCR product is type A, the rice being tested is or is a candidate rice variety with strong salt tolerance. If the PCR product is classified as G, then the rice being tested is or is a candidate rice variety with weak salt tolerance. In the PCR amplification system, the concentrations of the salt-tolerant allele-specific primers used to detect A nucleotide allele variations and the weak salt-tolerant allele-specific primers used to detect G nucleotide allele variations 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 method as described in any one of claims 1-2 in the cultivation of salt-tolerant rice, characterized in that, The method described in any one of claims 1-2 can be used to assist in determining the salt tolerance of rice during the rice cultivation process, and to select rice varieties with strong salt tolerance.

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