Rice high-yield salt-tolerant molecular marker and application thereof

Abstract

The invention belongs to the technical field of agricultural biology, 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 the 615th and 560th sites of chromosome 6 of a rice reference genome IRGSP1.0, and the polymorphic site is G / A. The invention further discloses a preparation method of the rice high-yield salt-tolerant molecular marker. The high-yield and salt-tolerant characteristics of the germplasm carrying the A nucleotide allelic variation at the site are obviously lower than those of the germplasm carrying the G nucleotide allelic variation, specifically, the yield of the GG genotype is the highest, the yield of the GA genotype is the middle, and the yield of the AA genotype is the lowest. By utilizing the molecular marker and the corresponding identification method, the genotype can be accurately and rapidly identified in the accurate design of the high-yield salt-tolerant rice, the phenotype identification cost of a field test in the later period is reduced, and the application of an accurate design technology of the rice in breeding is promoted.

Description

Technical Field

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

[0002] Rice is an important food crop in my country, and its high and stable yield plays an important role in ensuring my country's food security. Rice is a salt-sensitive crop and is often affected by salt damage, resulting in reduced yields. Therefore, cultivating new high-yield and salt-tolerant rice varieties is crucial to ensuring stable rice yields in areas susceptible to salt-alkali damage, and is also conducive to the development and utilization of saline-alkali land resources, increasing the planting area, and increasing food production.

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

[0004] 1. Genetic complexity: Salt tolerance in rice is a complex trait controlled by multiple genes, and its genetic mechanism is not yet fully understood. This means that in the breeding process, finding and combining multiple favorable genes is a difficult task, and the number of highly effective salt-tolerant genes discovered so far is limited.

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

[0006] 3. Low breeding efficiency: Traditional breeding methods are time-consuming and costly, and the screening process is complicated because the performance of salt tolerance traits is greatly affected by the environment. Although molecular marker-assisted selection and gene editing technologies have improved efficiency, the application of these technologies still needs to be improved and popularized.

[0007] 4. Balance between yield and quality: Salt-tolerant varieties often sacrifice a certain amount of yield or rice quality while improving salt tolerance. How to improve salt tolerance while maintaining high yield and quality is a difficult problem in breeding.

[0008] Therefore, the comprehensive use of multiple technologies to cultivate new rice varieties that are high-yield, salt-tolerant, high-quality, and highly adaptable is a major issue that urgently needs to be addressed in my country's agricultural production. Summary of the invention

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

[0010] The present invention provides a rice high-yield salt-tolerant molecular marker, which is located at position 615,560 of chromosome 6 of the 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, the primers are designed based on the flanking sequences of the molecular markers, 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 adopts the molecular marker detection method to obtain the result 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 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 a medium yield, and the AA genotype has the lowest yield.

[0020] Specifically, the primers are used for detection.

[0021] The specific method is as follows: extract the genomic DNA of the rice to be tested, use the genomic DNA as a template, and use 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, in the process of rice hybrid breeding, the method is used to assist in screening high-yield and salt-tolerant plants or varieties, and further obtains homozygous high-yield and salt-tolerant varieties through self-pollination or the like.

[0023] By using the molecular markers and corresponding identification methods disclosed in the present invention, genotypes can be accurately and quickly identified in the precise design of high-yield and salt-tolerant rice, the cost of phenotypic identification in later field trials can be reduced, and the application of rice precision design technology in breeding can be promoted. Through hybrid breeding, favorable alleles are introduced into the recipient variety, and molecular marker-assisted selection is used to select strains carrying favorable alleles in the hybrid population to create new high-yield and salt-tolerant materials. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Example 1: Location of loci related to high-yield and salt-tolerance in rice

[0026] (1) Phenotypic identification of salt tolerance in rice fields

[0027] More than 560 representative rice natural resources from major rice-growing areas in the world were selected to carry out field rice salt tolerance identification. The method is briefly described as follows: After soaking rice seeds at room temperature for 1 day, they are sown in the seedbed. When the seedlings are 4 weeks old, they are transplanted individually with a plant spacing of 15 cm and a row spacing of 25 cm. 15 days after transplanting, irrigate with salt water containing 0.3% salt until maturity. During this period, the salt content in the rice field is continuously measured, and the salt concentration in the rice field is maintained at 0.3-0.4% by adjusting the concentration of the irrigated salt water. After maturity, the individual plants are harvested, dried, threshed, and weighed. Each variety is repeated 10 times, and the average yield is finally calculated. Set 3 replicates.

[0028] A freshwater irrigation control was also set up, and the rest of the treatments were the same as the above salt treatments.

[0029] (2) Rice genotype identification

[0030] The rice genotype was identified by the second-generation high-throughput sequencing method. The process is as follows. 100 mg of rice leaves were taken, frozen in liquid nitrogen, ground, and genomic DNA was extracted by the CTAB method. After library construction, the sequencing was performed on the Illumina Novaseq platform to obtain the sequencing data Fastaq file, which was then aligned to the rice reference genome IRGSP1.0 using BWA. SNP / Indel variant sites were analyzed according to the GATK process to obtain rice genotype data.

[0031] (3) Genome-wide association study (GWAS) of loci related to rice high yield and salt tolerance

[0032] To identify the loci (QTLs) associated with high yield and salt tolerance in rice, we used the average yield of each rice variety under normal and salt stress as the phenotypic data, used the genotype data of SNP / Indel variant sites obtained by second-generation sequencing, and used the GEMMA software mixed linear model to conduct association analysis. Figure 1 As shown in Table 1, the molecular marker at position 615560 on chromosome 6 was found to be closely associated with the high-yield and salt-tolerant phenotype of rice, with a GWAS p value of 4.07×10 -8 The phenotypic contribution rate was 5.1%. Because beta was -4.18, the variant sequence A was negatively correlated with the high-yield and salt-tolerant traits.

[0033] Table 1. GWAS analysis of significant loci for high-yield and salt-tolerance in rice

[0034] chromosome Molecular markers Location 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 PCR primers for molecular marker identification of loci related to high-yield and salt-tolerance in rice

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

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

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

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

[0040] Using the above primers and KASP Master Mix, the following rice genomic DNA was used as a template, and KASP technology was used to carry out PCR amplification, fluorescence detection and data analysis on the LGC SNPline platform or ABI7500 / 7900 real-time quantitative fluorescence PCR instrument. The PCR program was: 94°C for 15 minutes; 94°C for 20 seconds, 58°C for 60 seconds, 10 cycles, and 0.6°C was reduced for each cycle; 94°C for 20 seconds, 55°C for 60 seconds, 30 cycles. After the reaction was completed, the FAM and HEX channel fluorescence signals were read. The FAM signal corresponded to the G genotype, and the HEX signal corresponded to the A genotype.

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

[0042] Table 2. Molecular marker m6_615KASP primers for rice genotype detection

[0043]

[0044]

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

[0046] The GWAS analysis results of Example 1 show that the molecular marker m6_615 genotype AA is negatively correlated with the high-yield salt-tolerant trait. 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 hybridized the rice variety Longdao (genotype AA) with the rice variety YHS (genotype GG), and the F2 population was propagated by single grain to obtain a recombinant inbred line population. In the F5 generation, the recombinant inbred line genotype was identified using the SNP molecular marker m6_615 according to the method of Example 2. Five strains were selected from the three genotypes of GG, GA, and AA, and the yield of rice under normal and salt stress was identified in the field according to the method of Example 1. The results are shown in Table 3. The GG genotype has the highest yield, the GA genotype has a medium yield, and the AA genotype has the lowest yield. This shows that the molecular marker m6_615 can be used to assist in the selection of high-yield salt-tolerant rice hybrid breeding, reduce the workload of field phenotypic identification, reduce costs, and improve breeding efficiency.

[0047] Table 3. Yield per plant of Longdao / YHS F5 lines with different genotypes under normal and salt stress

[0048] genotype Control single plant yield (g) Yield per plant 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 molecular marker for high-yield and salt-tolerant rice, characterized in that: It is located at position 615,560 on chromosome 6 of the rice reference genome IRGSP1.0, and the polymorphic site is G / A.

2. A primer for detecting the molecular marker as claimed in claim 1.

3. The primer according to claim 2, characterized in that It is a KASP primer.

4. The primer according to claim 2, characterized in that The primers are designed based on the flanking sequences of the molecular markers, and the specific flanking sequences are shown in SEQ ID NO: 1; More specifically, 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'.

5. Use of the molecular marker detection method as claimed in claim 1 in identifying high-yield and salt-tolerant rice varieties or plants.

6. The use according to claim 5, characterized in that The detection method is gene sequencing and molecular amplification.

7. 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 determine 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 a medium yield, and the AA genotype has the lowest yield.

8. The method according to claim 7, characterized in that The detection is performed using the primers described in any one of claims 2 to 4.

9. The method according to claim 8, characterized in that The specific method is as follows: extract the genomic DNA of the rice to be tested, use the genomic DNA as a template, and use the primers to perform PCR amplification to obtain a PCR product.

10. A molecular-assisted breeding method for high-yield salt-tolerant rice, characterized in that: In the process of rice hybrid breeding, the method as described in any one of claims 7 to 9 is used to assist in screening high-yield and salt-tolerant plants or varieties, and further obtain homozygous high-yield and salt-tolerant varieties through self-pollination or the like.

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