Rice high yield salt-tolerant molecular marker and application thereof

Abstract

The application belongs to the field of agricultural biotechnology, and relates to a rice high-yield salt-tolerant molecular marker and application thereof. The rice high-yield salt-tolerant molecular marker is located at 615,560 of rice reference genome IRGSP1.0 chromosome 6, and a polymorphic site is G / A. The high-yield salt-tolerant characteristics of the germplasm carrying A nucleotide allelic variation are significantly lower than those of the germplasm carrying G nucleotide allelic variation, specifically, the yield of the GG genotype is the highest, the yield of the GA genotype is in the middle, and the yield of the AA genotype is the lowest. The molecular marker and the corresponding identification method can accurately and rapidly identify the genotype in the precise design of the high-yield salt-tolerant rice, reduce the cost of phenotype identification in the later field test, and promote the application of the rice precise design technology in breeding.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural biotechnology and relates to a high-yield salt-tolerant molecular marker of rice and application thereof. BACKGROUND

[0002] Rice is an important food crop in China, and its high and stable yield plays an important role in ensuring food security in China. Rice is a salt-sensitive crop and is often affected by salt damage, resulting in yield reduction. Therefore, breeding new salt-tolerant rice varieties is crucial for ensuring the stable yield of rice in areas prone to salt and alkali hazards, and is also conducive to the development and utilization of saline-alkali land resources, the increase of planting area and the improvement of grain yield.

[0003] However, the breeding of salt-tolerant rice in China faces many challenges, mainly in the following aspects.

[0004] 1. Genetic complexity: The salt tolerance of rice is a complex trait controlled by multiple genes, and its genetic mechanism is not fully understood. This means that it is a difficult task to find and combine multiple beneficial genes in the breeding process, and the number of high-efficiency salt-tolerant genes discovered is limited.

[0005] 2. Environmental adaptability: The salt composition, pH value and soil texture of different saline-alkali lands differ greatly, making it difficult for a single salt-tolerant variety to adapt to all types of saline-alkali lands, and it is necessary to develop varieties that can adapt to multiple environments.

[0006] 3. Low breeding efficiency: Traditional breeding methods are time-consuming and costly, and the selection process is complex due to the strong environmental influence on the expression of salt tolerance traits. Although molecular marker-assisted selection and gene editing techniques have improved efficiency, the application of these techniques still needs to be improved and popularized.

[0007] 4. Yield and quality balance: Salt-tolerant varieties often sacrifice certain yield or rice quality while improving salt tolerance. How to maintain high yield and quality while improving salt tolerance is a difficult problem in breeding.

[0008] Therefore, the comprehensive use of multiple technologies to breed new rice varieties with high yield, salt tolerance, high quality and high adaptability is a major issue that needs to be addressed in China's agricultural production. SUMMARY

[0009] The purpose of the present application is to disclose a method for designing high-yield salt-tolerant rice, and the application of the method in creating new salt-tolerant rice varieties.

[0010] The present application provides a high-yield salt-tolerant molecular marker of rice, which is located at position 615,560 of chromosome 6 of rice reference genome IRGSP1.0, and the polymorphic site is G / A.

[0011] The present invention provides primers for detecting the molecular markers, specifically, KASP primers.

[0012] More specifically, primers are designed based on the flanking sequences of the molecular marker, and the specific flanking sequences are shown in SEQ ID NO: 1;

[0013] More specifically, the KASP primers are as follows:

[0014] Allele G-specific primer: 5′-GAAGGTGACCAAGTTCATGCTtacatcggcgtcgttcaagG-3′;

[0015] Allele A-specific primer: 5′-GAAGGTCGGAGTCAACGGATTtacatcggcgtcgttcaagA-3′;

[0016] Universal primer: 5′-acagagacatacgataaagaggtcg-3′.

[0017] The present invention provides application of the molecular marker detection method in identifying high-yield and salt-tolerant rice varieties or plants.

[0018] Specifically, the detection method is gene sequencing and molecular amplification.

[0019] The present invention provides a method for identifying high-yield and salt-tolerant rice varieties or plants, which uses the molecular marker detection method to obtain the molecular marker results to judge the high-yield and salt-tolerant rice, wherein the high-yield and salt-tolerant characteristics of the germplasm carrying the A nucleotide allele at this site are significantly lower than those of the germplasm carrying the G nucleotide allele, specifically, the GG genotype has the highest yield, the GA genotype has an intermediate yield, and the AA genotype has the lowest yield.

[0020] Specifically, the detection is performed using the primers.

[0021] The specific method is as follows: extracting genomic DNA from the rice to be tested, using the genomic DNA as a template and adopting the primers to perform PCR amplification to obtain a PCR product.

[0022] The present invention provides a molecular-assisted breeding method for high-yield and salt-tolerant rice, which is characterized in that, during the rice hybrid breeding process, the method is used to assist in screening high-yield and salt-tolerant plants or varieties, and further homozygous high-yield and salt-tolerant varieties are obtained through self-pollination or the like.

[0023] The molecular marker and the corresponding identification method disclosed by the application can accurately and rapidly identify genotypes in high-yield salt-tolerant rice precise design, reduce the cost of phenotype identification in later field tests, and promote the application of rice precise design technology in breeding. Through hybridization breeding, favorable allelic genotypes are introduced into the receptor variety, and molecular marker assisted selection is used to select the lines carrying favorable allelic genotypes in the hybrid population, and new high-yield salt-tolerant materials are created. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The results of whole genome association analysis (GWAS) of rice yield under normal and salt stress. The horizontal dashed line represents the significance threshold p=10 -5 , and the arrow position represents the position of the SNP molecular marker m6_615. DETAILED DESCRIPTION

[0025] Example 1: Positioning of rice high-yield salt-tolerant related sites

[0026] (1) Field salt tolerance phenotype identification of rice

[0027] More than 560 rice natural resources from representative rice planting areas around the world were selected to carry out field salt tolerance identification of rice. The method is briefly described as follows: after soaking the rice seeds at room temperature for 1 day, the seeds were sown in a seedbed, and the seedlings were transplanted at an age of 4 weeks with a plant spacing of 15 cm and a row spacing of 25 cm. After 15 days of transplanting, salt water containing 0.3% salt was irrigated until maturity. The salt content in the rice field was continuously measured during the period, and the salt concentration in the rice field was maintained at 0.3-0.4% by adjusting the salt concentration of the irrigated salt water. After maturity, the single plant was harvested, dried, threshed, and weighed. Ten replicates were calculated for each variety, and finally the yield average was calculated. Three replicates were set.

[0028] A fresh water irrigation control was also set up, and the rest of the treatment was the same as the above salt treatment.

[0029] (2) Genotype identification of rice

[0030] The genotype of rice was identified by a second-generation high-throughput sequencing method, and the process was as follows. 100 mg of rice leaves were taken, frozen in liquid nitrogen, ground, and the genomic DNA was extracted by the CTAB method. After library construction, sequencing was performed on the Illumina Novaseq platform, and sequencing data Fastaq files were obtained. Then, BWA was used to align to the rice reference genome IRGSP1.0. According to the GATK process, SNP / Indel variation sites were analyzed, and thus the rice genotype data was obtained.

[0031] (3) Whole genome association analysis (GWAS) of rice high-yield salt-tolerant related sites

[0032] To identify the QTL of high yield and salt tolerance in rice, we used the average yield of each rice variety under normal and salt stress as the phenotype data, and the SNP / Indel variation site genotype data obtained by second-generation sequencing, and used the GEMMA software mixed linear model to carry out association analysis. The results are shown in Figure 1 and Table 1, it is found that the molecular marker at position 615560 of chromosome 6 is closely related to the high yield and salt tolerance phenotype of rice, and the GWAS p value is 4.07x10 -8 , and the phenotype contribution rate is 5.1%. Because beta is -4.18, the variation sequence A is negatively related to the high yield and salt tolerance trait.

[0033] Table 1, GWAS analysis of significant sites of high yield and salt tolerance in rice

[0034] Chromosome Molecular marker Position Variant sequence Reference sequence beta p-value Phenotypic variance 6 m6_615 615,560 A G -4.18044 4.07E-08 0.050841366

[0035] Example 2: Development of molecular marker PCR identification primer of rice high yield and salt tolerance related site

[0036] The molecular marker m6_615 is located at position 615,560 of chromosome 6 of the rice reference genome IRGSP1.0 6, and the polymorphic site is G / A. The 200bp flanking sequence (SEQ ID NO: 1) thereof is obtained from the genome group, and the competitive allele-specific PCR (KASP) primer is designed by Primer3 as follows:

[0037] Allele site G specific primer: 5'-GAAGGTGACCAAGTTCATGCTtacatcggcgtcgttcaagG-3';

[0038] Allele site A specific primer: 5'-GAAGGTCGGAGTCAACGGATTtacatcggcgtcgttcaagA-3';

[0039] Universal primer: 5'-acagagacatacgataaagaggtcg-3'.

[0040] Using the above primer and KASP Master Mix, the following rice genomic DNA as a template, using KASP technology in LGC SNPline platform, or ABI7500 / 7900 real-time quantitative fluorescence PCR instrument to carry out PCR amplification and fluorescence detection and data analysis. The PCR program is: 94℃ for 15 minutes; 94℃ for 20 seconds, 58℃ for 60 seconds, 10 cycles, each cycle decreases by 0.6℃; 94℃ for 20 seconds, 55℃ for 60 seconds, 30 cycles. After the reaction is completed, read the FAM and HEX channel fluorescence signal, FAM signal corresponds to G genotype, HEX signal corresponds to A genotype.

[0041] Using the above method, we detected the genotypes of 37 rice varieties at the locus of molecular marker m6_615, and the results are shown in Table 2.

[0042] Table 2. Genotypes of rice detected by KASP primers of molecular marker m6_615

[0043]

[0044]

[0045] Example 3: Application of molecular marker m6_615 in the creation of high-yield salt-tolerant rice lines

[0046] The GWAS analysis results of Example 1 show that the genotype AA of molecular marker m6_615 is negatively correlated with the traits of high yield and salt tolerance. In order to verify whether the molecular marker m6_615 can be used for molecular marker-assisted selection in the creation of high-yield salt-tolerant rice lines, we crossed the rice variety Longdao (genotype AA) with the rice variety YHS (genotype GG), and obtained a recombinant inbred line population by single-seed descent of the F2 population. The genotypes of the recombinant inbred lines were identified by SNP molecular marker m6_615 in the F5 generation according to the method of Example 2. Five strains of each of the GG, GA, and AA genotypes were selected and their yields under normal and salt stress were identified in the field according to the method of Example 1. The results are shown in Table 3. The yield of the GG genotype was the highest, the yield of the GA genotype was in the middle, and the yield of the AA genotype was the lowest. This shows that the molecular marker m6_615 can be used for molecular marker-assisted selection in the hybridization breeding of high-yield salt-tolerant rice, reducing the workload of field phenotypic identification, reducing costs, and improving breeding efficiency.

[0047] Table 3. Yields of Longdao / YHS F5 strains with different genotypes under normal and salt stress

[0048] Genotype Control plant yield (g) Plant yield under salt stress (g) GG 28.8 23.6 GG 30.9 23.5 GG 30.2 22.6 GG 28.9 22 GG 29.4 23.6 GA 27.2 21.9 GA 29.2 22 GA 26.9 22.9 GA 27.5 21.4 GA 28.2 23.2 AA 24.6 20.3 AA 25.8 19.7 AA 25.2 20.7 AA 23.4 19.6 AA 25.6 20.1

Claims

1. A method for detecting a high-yield and salt-tolerant rice molecular marker for use in identifying high-yield and salt-tolerant rice varieties or plants, wherein the molecular marker is located at position 615,560 on chromosome 6 of the rice reference genome IRGSP1.0, and the polymorphic site is G / A.

2. The use according to claim 1, characterized in that The detection method is gene sequencing or molecular amplification.

3. A method for identifying high-yield and salt-tolerant rice varieties or plants, characterized in that: The molecular marker detection method as described in claim 1 is used to obtain the results of the molecular marker to judge the high-yield and salt-tolerant rice, wherein the high-yield and salt-tolerant characteristics of the germplasm carrying the A nucleotide allele at the polymorphic site are significantly lower than those of the germplasm carrying the G nucleotide allele, the GG genotype has the highest yield, the GA genotype has a medium yield, and the AA genotype has the lowest yield.

4. The method according to claim 3, wherein The method uses primers for detecting the molecular marker as claimed in claim 1 for detection.

5. The method according to claim 4, wherein The primer is a KASP primer.

6. The method according to claim 5, wherein The KASP primers are as follows: Allele G-specific primer: 5′-GAAGGTGACCAAGTTCATGCTtacatcggcgtcgttcaagG-3′; Allele A-specific primer: 5′-GAAGGTCGGAGTCAACGGATTtacatcggcgtcgttcaagA-3′; Universal primer: 5′-acagagacatacgataaagaggtcg-3′.

7. The method according to any one of claims 4 to 6, characterized in that The specific method is as follows: extracting genomic DNA from the rice to be tested, using the genomic DNA as a template and adopting the primers to perform PCR amplification to obtain a PCR product.

8. A molecular-assisted breeding method for high-yield and salt-tolerant rice, characterized in that: In the process of rice hybrid breeding, the method according to any one of claims 3 to 7 is used to assist in screening high-yield and salt-tolerant plants or varieties, and further self-pollination is used to obtain homozygous high-yield and salt-tolerant varieties.

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