A molecular marker method for QTL ALT5 in rice salt-alkali tolerance

Abstract

The invention discloses a molecular marker method for rice salt-alkali tolerance QTL ALT5, which belongs to the field of genetic engineering. The rice genome to be tested with Longdao No. 5 ancestry is used as a template, and the primers of the nucleotide sequences shown in SEQ ID No. 1 and SEQ ID No. 2 are used for PCR amplification. If a 175bp product can be amplified, the rice to be tested carries the rice salt-alkali tolerance QTL ALT5. The invention uses the cold-region salt-alkali tolerant rice "Longdao No. 5" as a parent, and improves the salt-alkali tolerance of the high-yield japonica rice variety "Dongnong 423" with indica rice ancestry. In the F6 generation segregation population, a salt-alkali tolerance QTL ALT5 from "Longdao No. 5" is located on the fifth chromosome, and a PCR practical and economical marker ALT5-1 closely linked to the salt-alkali tolerance QTL is identified, which can be used to effectively carry out molecular assisted selection breeding research on rice salt-alkali tolerance.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a molecular marker method for rice salt-alkali tolerance QTL ALT5. Background Art

[0002] Soil salinization is one of the most serious abiotic stresses and is considered an important environmental limiting factor affecting global agricultural ecosystems and food security. It has many negative effects on crops, including osmotic pressure, high pH stress, disruption of ion balance, limited nutrient absorption, and inhibition of plant growth, development, and yield. According to the Food and Agriculture Organization of the United Nations (FAO), the total area of ​​saline-alkali land in the world currently exceeds 1 billion hectares. It is estimated that by 2050, more than 50% of cultivated land will be salinized due to low rainfall, high surface evaporation, unreasonable irrigation, poor farming methods, excessive use of fertilizers and other similar reasons. China is the third largest country in the world with saline-alkali soil, with a total area of ​​9.91×10 7 Hectares, mainly distributed in Northwest, North China, Northeast China and coastal areas. The Songnen Plain in Northeast China is the world's largest alkaline soil distribution area, and the main component of salt is carbonate. The soil pH in this area is above 8.5, and the pH of highly alkaline soil reaches 9.0-10.5. A major problem in the region is alkali damage caused by large amounts of sodium carbonate (Na2CO3) or sodium bicarbonate (NaHCO3). Research and practice have shown that growing rice on saline-alkali land is one of the best ways to improve saline-alkali land, increase farmers' income, agricultural benefits and ecological environment. Therefore, cultivating salt-alkali tolerant varieties is an important goal of japonica rice breeding in the saline-alkali land area of ​​Northeast China. Traditional breeding methods are time-consuming, labor-intensive, difficult to identify phenotypes, and have low breeding efficiency. Since the salt-alkali tolerance of rice is a typical quantitative trait, involving multiple gene loci, and there is an interaction effect with the environment, it is difficult to genetically improve salt-alkali tolerance. Using salt-alkali tolerance genes and molecular markers closely linked to them, molecular marker-assisted selection breeding can effectively solve this problem.

[0003] So far, some salt-alkali tolerance gene loci have been located, and some linkage marker information has been reported, but the exchange rate is high and the selection effect is poor. At the same time, a few tightly linked markers belong to SNP information obtained by sequencing and have not been converted into tightly linked markers. Therefore, auxiliary molecular markers for salt-alkali tolerance gene loci are still scarce. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a molecular marker method for QTL ALT5 of rice salt-alkali tolerance.

[0005] The technical scheme of the present invention is: a molecular marker method for rice salt-alkali tolerance QTL ALT5, using the genome of the rice to be tested with Longdao No. 5 as a template, using primers of the nucleotide sequences shown in SEQ ID No. 1 and SEQ ID No. 2 for PCR amplification, if a 175bp product can be amplified, the rice to be tested carries the rice salt-alkali tolerance QTL ALT5.

[0006] A method for breeding salt-alkali tolerant rice using molecular markers tightly linked to the salt-alkali tolerance QTL ALT5 of rice, wherein Longdao 5 or a blood material of Longdao 5 carrying the salt-alkali tolerance QTL ALT5 is used as a parent to hybridize with a rice variety to be improved, and PCR amplification is performed on the hybrid material using primers of the nucleotide sequences shown in SEQ ID No.1 and SEQ ID No.2. If a product of 175 bp can be amplified, the hybrid material is a material into which the salt-alkali tolerance QTL ALT5 is introduced and has salt-alkali tolerance.

[0007] A kit contains primers of nucleotide sequences shown in SEQ ID No.1 and SEQ ID No.2.

[0008] The kit is used for molecular markers of rice salt-alkali tolerance QTL ALT5 and breeding of salt-alkali tolerant rice.

[0009] The present invention uses the cold-region salt-alkali tolerant rice "Longdao No. 5" as a parent, improves the salt-alkali tolerance of the high-yield japonica rice variety "Dongnong 423" with indica rice ancestry, and performs auxiliary selection on the salt-alkali tolerance QTL ALT5 in the offspring population. In the F6 generation segregation population, a salt-alkali tolerance QTL ALT5 from "Longdao No. 5" is located on chromosome 5. The QTL simultaneously controls the salt-alkali tolerance index, seedling height, seedling dry weight and root dry weight under saline-alkali conditions, and identifies a practical and economical PCR marker ALT5-1 that is closely linked to the salt-alkali tolerance QTL, which can be used to effectively carry out molecular assisted selection breeding research on cold tolerance in rice during the booting period.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] 1. The present invention identified the salt-alkali tolerance QTL ALT5 on chromosome 5 and the co-dominant molecular marker ALT5-1 for genotyping thereof.

[0012] 2. Through the auxiliary screening of the cold-tolerant QTL ALT5 marker at the booting stage, rice varieties or materials with significantly improved cold-tolerant ability at the booting stage can be obtained.

[0013] 3. The molecular markers of the present invention can be used for the salt-alkali tolerance genotype selection of the hybrid offspring of "Longdao No. 5" as the parent and the hybrid offspring of related derivative materials, and can effectively identify the cold-resistant materials with the QTL, facilitate timely hybridization and breeding, improve breeding efficiency, and accelerate the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the detection results of the ALT5 molecular marker in an embodiment of the present invention; the markings in the figure are as follows: M: molecular marker; 1: Donnong 423; 2: Longdao No. 5; 3-12: F6 rice strains with weak salt-alkali tolerance; 13-22: F6 rice strains with strong salt-alkali tolerance. DETAILED DESCRIPTION

[0015] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0016] The biological materials "Longdao No. 5" and "Dongnong 423" used in the present invention are both commercial conventional varieties, and those skilled in the art can purchase them from the market.

[0017] 1. Discovery and molecular markers of QTL ALT5 for cold tolerance at the booting stage in rice

[0018] "Longdao No. 5" is a representative cold-region rice germplasm resource with strong salt-alkali tolerance. The salt-alkali-tolerant "Longdao No. 5" is used as the male parent and the high-yield and disease-resistant variety "Dongnong 423" is used as the female parent for hybridization, and the F6 generation segregation population is obtained through single-grain transmission. In 2022-2023, this population was used for QTL positioning analysis of salt-alkali tolerance traits at the seedling stage. A stable inherited major effect QTL-ALT5 was detected between the short arm ends of rice chromosome 5. Its synergistic allele came from the salt-alkali-tolerant resource "Longdao No. 5". A molecular marker ALT5-1 (nucleotide sequence shown in SEQ ID No. 1 and SEQ ID No. 2) closely linked to the salt-alkali tolerance QTL ALT5 was detected in this interval. The introduction of the ALT5 gene can increase the salt-alkali tolerance level of rice by 2 to 3 levels. In the F6 generation population, the salt-alkali tolerance level of the strains carrying this QTL is 1 to 2, and the salt-alkali tolerance level of the strains without this QTL is 5 to 7.

[0019] 2. Application of ALT5 molecular assisted breeding in rice

[0020] The molecular marker ALT5-1 was applied to the F6 population of the breeding progeny of Longdao 5 and Dongnong 423 for verification:

[0021] 1) DNA was extracted using the conventional CTAB method.

[0022] 2) The PCR amplification system was 15 μL, containing 50 mM KCl, 10 mM Tris-HCl (pH 8.8), 0.1% Triton-X, 1.5 mM MgCl2, 200 μM each of dNTPs, 0.2 μM of each primer and 0.5 U Taq DNA polymerase.

[0023] The reaction procedure was pre-denaturation at 94°C for 5 min; each cycle consisted of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 2 min, for 30 cycles; and finally extension at 72°C for 8 min.

[0024] 3) The PCR amplification products were separated by 2.5% agarose gel electrophoresis.

[0025] 4) Observe and record the results after staining.

[0026] The results are as follows Figure 1 As shown, the offspring materials carrying the salt-alkali tolerance QTL ALT5 (salt-alkali tolerance level 1-2) can amplify a band of about 175 bp, while the offspring without the salt-alkali tolerance QTL ALT5 (salt-alkali tolerance level 5-7) can amplify a band of about 207 bp. It can be seen that this molecular marker can be used to identify whether the material to be tested contains the salt-alkali tolerance QTL ALT5.

Claims

1. A method for detecting QTLs for salt-alkali tolerance in rice ALT5 The molecular labeling method is characterized in that The hybrid offspring of Longdao 5 and Dongnong 423 was used as the test rice, and the test rice genome was used as a template. PCR amplification was performed using primers of the nucleotide sequences shown in SEQ ID No. 1 and SEQ ID No.

2. If a 175 bp product can be amplified, the test rice carries the rice salt-alkali tolerance QTL ALT5 .

2. Utilizing QTLs related to rice salt-alkali tolerance ALT5 A method for breeding salt-alkali tolerant rice using tightly linked molecular markers, characterized in that: The hybrid offspring of Longdao No. 5 and Dongnong 423 were used as the breeding material, and the primers of the nucleotide sequences shown in SEQ ID No. 1 and SEQ ID No. 2 were used for PCR amplification of the hybrid offspring material. If a product of 175 bp could be amplified, the hybrid offspring material was introduced with the salt-alkali tolerance QTL. ALT5 The material is salt-alkali resistant.

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