RGG2 gene molecular marker related to rice grain weight and yield and application of RGG2 gene molecular marker

Abstract

The invention discloses an RGG2 gene molecular marker related to rice grain weight and yield and application of the RGG2 gene molecular marker, the rice RGG2 gene molecular marker is an RGG2-Hap6 haplotype in RGG2 gene haplotypes, and the haplotype is that three basic groups of GGA are inserted into the 68th basic group of RGG2-Hap1. According to the marker primer and the identification method designed by the invention, the RGG2-Hap6 haplotypes can be simply and rapidly distinguished, and genotype selection is carried out. By combining the designed molecular marker, the excellent allelic variation RGG2-Hap6 is introduced into a plurality of rice varieties through hybridization and backcross methods, the rice grain weight and the single plant yield can be improved, and potential breeding utilization value is achieved.

Description

Technical Field

[0001] The present invention relates to an RGG2 gene molecular marker related to rice grain weight and yield and its application, belonging to the field of plant molecular biology. Background Art

[0002] Rice is one of the most important food crops in the world, providing the main food source for more than half of the world's population. With the growth of the population and the continuous reduction of arable land area, the improvement of rice yield has increasingly become the focus of attention. Rice yield is a complex trait, mainly determined by the number of panicles per plant, the number of grains per panicle, and the 1000-grain weight, etc. Grain weight is an important part of yield, determined by grain shape and grain plumpness. At present, multiple grain shape-related QTLs in rice have been mapped and cloned, and the regulatory mechanism has also been initially understood. However, due to gene pleiotropy, while increasing yield, other agronomic traits will also change. Therefore, most genes related to yield cannot be directly applied to rice breeding. In the process of high-yield rice breeding, it is necessary to screen genes that can regulate yield but have no or little effect on other agronomic traits for improving rice yield traits.

[0003] The heterotrimeric G protein (G protein) signaling pathway is a very important pathway for regulating grain weight. G protein is a multifunctional component of the transmembrane signal transduction pathway, related to a wide range of growth and developmental responses in plants, fungi, and animals. In the rice genome, 1 Gα gene (RGA1), 1 Gβ gene (RGB1), and 5 Gγ homologous genes (RGG1, RGG2, GS3, qPE9-1 / DEP1, and GGC2) have been identified. Multiple G protein genes have been proven to be involved in regulating rice grain size and yield. Among them, GS3 has been determined to be a major QTL regulating grain length and grain weight, and the gs3 allele will lead to an increase in grain length and grain weight; qPE9-1 regulates traits such as panicle length, grain length, and grain weight, thereby affecting rice yield; the rice RGG2 gene negatively regulates rice grain weight and yield. Specifically, overexpression of the RGG2 gene results in short and round grains of the plant, reduced grain weight, and decreased yield per plant, while the phenotype of the knockout mutant is opposite. There are 6 different haplotypes of the RGG2 gene, and the RGG2-Hap6 haplotype is only found in the large-grain variety Suyunuo and the local germplasm resource large-grain japonica, but its utilization value in high-yield rice breeding is still unclear. Summary of the Invention

[0004] Objective of the Invention: The objective of the present invention is to provide an RGG2 gene molecular marker related to rice grain weight and yield and its application. In this application, an excellent haplotype RGG2-Hap6 of the RGG2 gene closely associated with rice grain weight and yield per plant is identified, corresponding molecular markers are developed, the effect of RGG2-Hap6 in increasing rice grain weight and yield per plant is clarified, providing strong technical support for high-yield rice breeding and genetic improvement.

[0005] Technical Solution: The present invention provides the application of a rice RGG2 gene molecular marker in increasing rice grain weight and yield. The rice RGG2 gene molecular marker is the RGG2-Hap6 haplotype in the RGG2 gene haplotypes, and this haplotype inserts three bases GGA at the 68th base of RGG2-Hap1.

[0006] Furthermore, the amino acid sequence of the RGG2-Hap6 haplotype is as shown in SEQ ID NO.4, and the nucleotide sequence is as shown in SEQ ID NO.3.

[0007] The present invention also provides a molecular marker primer for detecting RGG2 gene haplotypes. The molecular marker primer includes a common forward primer RGG2-1F with a nucleotide sequence as shown in SEQ ID NO.5 and two reverse downstream primers RGG2-1R and RGG2-2R with nucleotide sequences as shown in SEQ ID NO.6 and SEQ ID NO.7.

[0008] The present invention also provides a kit, which contains the above-mentioned molecular marker primers.

[0009] The present invention also provides a method for identifying RGG2 gene haplotypes, including the following steps: extracting the genomic DNA of the rice to be tested; using the extracted rice genomic DNA as a template and performing PCR amplification with the above-mentioned molecular marker primers.

[0010] Furthermore, if the primer pair RGG2-1F and RGG2-1R can amplify a characteristic band of 495bp, and the primer pair RGG2-1F and RGG2-2R cannot amplify a characteristic band, then the rice material carries the RGG2-Hap1 allele; if the primer pair RGG2-1F and RGG2-2R amplifies a characteristic band of 498bp, and the primer pair RGG2-1F and RGG2-1R cannot amplify a characteristic band, then the rice material carries the RGG2-Hap6 allele; if the primer pair RGG2-1F and RGG2-1R can amplify 495bp, and the primer pair RGG2-1F and RGG2-2R amplifies a characteristic band of 498bp, then the rice material carries both the RGG2-Hap1 and RGG2-Hap6 alleles.

[0011] Furthermore, the PCR amplification system includes molecular marker primers, 2×Taq PCR Master Mix (Novoprotein) and double distilled water.

[0012] Furthermore, the PCR amplification program is pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min.

[0013] The present invention also provides a method for cultivating high-yield rice, characterized in that RGG2-Hap6 is introduced into rice through hybridization and backcrossing until a rice strain with significantly increased grain weight and single-plant yield is obtained.

[0014] Furthermore, the primers used to identify and screen rice plants containing the RGG2-Hap6 allele in hybrid and backcross progenies are the above-mentioned molecular marker primers.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1) The present invention provides an application of rice RGG2 gene in cultivating high-yield rice, and discloses an excellent haplotype RGG2-Hap6 for increasing rice yield.

[0016] 2) The present invention provides a molecular marker for detecting the RGG2 gene, which can effectively and accurately identify the genotype of the rice RGG2 gene. The molecular marker is a practical and economical marker based on PCR, which is easy to operate, does not require expensive equipment, has good primer specificity, and has high amplification efficiency.

[0017] 3) The molecular markers of the present invention are used for molecular marker-assisted breeding of the RGG2-Hap6 gene, which can realize rapid, simple and efficient rice genotype identification, save years of multi-point phenotypic identification or high-cost gene analysis and sequencing, reduce breeding costs, and improve breeding efficiency and rice quality and yield.

[0018] 4) The present invention provides a breeding method for cultivating high-yield rice, which can achieve directional improvement and breeding of yield of different rice varieties, and has good practical potential and promotion value.

[0019] Figure Description

[0020] Figure 1 The sequencing peak diagram (a) and amino acid sequence comparison diagram (b) of different haplotypes of RGG2, RGG2-Hap1 and RGG2-Hap6, and the grain type comparison diagram of Zhennuo 19 and Zhennuo 20 containing RGG2-Hap1 and Suyu Nuo containing RGG2-Hap6 (c);

[0021] Figure 2Electrophoresis results of PCR amplification products of different primer combinations of the present invention on 1% agarose gel;

[0022] Figure 3 Sequencing peak diagrams of amplification products of different rice varieties using primers RGG2-1F / RGG2-1R and RGG2-2F / RGG2-2R;

[0023] Figure 4 For Zhennuo 19 and Zhennuo 19 RGG2-Hap6 Comparison of plant type, grain type and yield per plant of introgression lines;

[0024] Figure 5 For Zhennuo 20 and Zhennuo 20 RGG2-Hap6 Comparison of plant type, grain type and yield per plant of introgression lines. Detailed implementation manners

[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0026] Example 1 Design of molecular marker primers for identifying RGG2-Hap6 genotype

[0027] There are a total of 6 haplotypes of RGG2, namely Hap1 to Hap6. As Figure 1 shown, the RGG2-Hap6 haplotype (nucleotide sequence as shown in SEQ ID NO.3; amino acid sequence as shown in SEQ ID NO.4) has an insertion of three bases GGA at the 68th base of RGG2-Hap1 (nucleotide sequence as shown in SEQ ID NO.1; amino acid sequence as shown in SEQ ID NO.2). This haplotype was only amplified in the large-grain variety Suyunuo and the local germplasm resource large-grain japonica. Therefore, it is inferred that it may be an excellent haplotype for improving rice yield.

[0028] 1. Primer design

[0029] Taking the variable sites of RGG2-Hap1 and RGG2-Hap6 as the 3' ends, and introducing a strong mismatch base A to replace the original base G at the variable sites, the downstream PCR amplification primers RGG2-1R and RGG2-2R are obtained, and the primer sequences are as follows:

[0030] RGG2-1R: CCACCTCCTCCTCCTCCTCtG (amplifying RGG2-Hap1)

[0031] RGG2-2R: CCACCTCCTCCTCCTCCTCCTCtG (amplifying RGG2-Hap6)

[0032] Using the PRIMER5.0 software, a common upstream primer is designed, and the primer sequence is as follows:

[0033] RGG2-1F: AGGCAAGTGTTACCAAA

[0034] All primers were synthesized by Nanjing Qingke Biotechnology Co., Ltd.

[0035] 2. Rice DNA extraction

[0036] The DNA of rice leaves such as Suyunuo, Zhennuo 19, Zhennuo 20, Nanjing 9108, and Huaidao 5 was extracted using the CTAB method. Take about 1 g of rice leaves and place them in a centrifuge tube. After quick freezing with liquid nitrogen, grind the sample thoroughly in a grinder; add 600 μL of preheated CTAB (cetyltrimethylammonium bromide), heat in an oven at 65 °C for 1 h, mix well every 20 min, add 600 μL of chloroform, and mix well; centrifuge at 12000 rpm for 10 min, pipette 500 μL of the supernatant into a new 1.5 mL centrifuge tube, add an equal volume of isopropanol to each, invert and mix well for 15 s, then place in a -20 °C refrigerator for 30 min; centrifuge at 12000 rpm for 10 min, discard the supernatant to leave a white precipitate, add 1 mL of 70% ethanol to the precipitate for washing, and repeat the washing twice; place the centrifuge tube in a laminar flow hood to air dry for 30 min, add 300 ddH2O to dissolve and mix well, and place in a -20 °C refrigerator for standby.

[0037] 3. PCR amplification

[0038] PCR amplification was carried out according to the following reaction system:

[0039] Table 1

[0040]

[0041] The PCR reaction conditions were: pre-denaturation at 95 °C for 5 min; then denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s, extension at 72 °C for 45 s, for 35 cycles; finally, extension at 72 °C for 5 min to end the reaction.

[0042] The above amplification products were separated and detected by agarose gel electrophoresis with a concentration of 1% to clarify the haplotypes of the RGG2 gene in rice varieties. The results are as Figure 2 shown. Using the specific primer pair combination RGG2-1F and RGG2-1R, a band with a size of 495 bp could be amplified in Zhennuo 19, Zhennuo 20, Nanjing 9108, and Huaidao 5, while no characteristic band could be amplified in Suyunuo containing the RGG2-Hap6 haplotype; using the specific primer pair combination RGG2-1F and RGG2-2R, a band with a size of 498 bp could be amplified in Suyunuo containing the RGG2-Hap6 haplotype, while it could not be amplified in Zhennuo 19, Zhennuo 20, Nanjing 9108, and Huaidao 5.

[0043] By the above method, it is possible to quickly distinguish whether the rice material contains the RGG2-Hap6 allele. If the primer pair combination RGG2-1F and RGG2-1R can amplify a characteristic band of 495 bp in size, while the primer pair combination RGG2-1F and RGG2-2R cannot amplify a band of 498 bp in size, then this rice material does not contain the RGG2-Hap6 allele; if the primer pair combination RGG2-1F and RGG2-1R cannot amplify a band of 495 bp in size, while the primer pair combination RGG2-1F and RGG2-2R can amplify a band of 498 bp in size, then this rice material contains the RGG2-Hap6 allele; if the primer pair combination RGG2-1F and RGG2-1R and the primer pair combination RGG2-1F and RGG2-2R can respectively amplify characteristic bands of 495 bp and 498 bp in size, then this rice material contains the heterozygous RGG2-Hap6 allele.

[0044] 4. Verification of method feasibility

[0045] Send the above amplification products to Nanjing Tsingke Biotechnology Co., Ltd. for sequencing. Compare and analyze the sequencing results to determine whether the amplification products are the RGG2 gene.

[0046] The results are as Figure 3 shown. The amplification products of the primer pair combination RGG2-1F and RGG2-1R in Huaidao 5, Nanjingjing 9108, Zhennuo 19 and Zhennuo 20, and the amplification products of the primer pair combination RGG2-1F and RGG2-2R in Suyunuo are all the RGG2 gene. This indicates that the above primer combination of the RGG2-Hap6 gene molecular marker is accurate and feasible.

[0047] Example 2 A breeding method for cultivating high-yield rice

[0048] In this example, taking the rice varieties Zhennuo 19 and Zhennuo 20 as examples, after introducing the RGG2-Hap6 haplotype in Suyunuo, the grain weight and per-plant yield of rice can be increased. The specific implementation method is as follows:

[0049] 1. Introduction of the RGG2-Hap6 haplotype into different rice varieties

[0050] 1.1 Cross the rice varieties Zhennuo 19 and Zhennuo 20 with Suyunuo containing the RGG2-Hap6 haplotype respectively to obtain F1 hybrid seeds.

[0051] 1.2 Use the molecular marker primers of Example 2 to detect the F1 generation, select the rice plants containing the RGG2-Hap6 haplotype and backcross them with Zhennuo 19 and Zhennuo 20 respectively to obtain the BC1F1 generation.

[0052] 1.3. Similar to step 1.2, select the rice plants with the RGG2-Hap6 haplotype in the BC1F1 generation and backcross them with Zhennuo 19 and Zhennuo 20 respectively to obtain the BC2F1 generation. Then, select the rice plants with the RGG2-Hap6 haplotype in the BC2F1 generation and backcross them with Zhennuo 19 and Zhennuo 20 respectively to obtain the BC3F1 generation. Then, select the rice plants with the RGG2-Hap6 haplotype in the BC3F1 generation and backcross them with Zhennuo 19 and Zhennuo 20 respectively to obtain the BC4F1 generation.

[0053] 1.4. Plant the BC4F1 generation into plant lines, screen the rice plants with the RGG2-Hap6 haplotype, and self-cross to obtain BC4F2 rice seeds. Plant the BC4F2 generation into plant lines, screen the rice plants with the RGG2-Hap6 haplotype, and self-cross to obtain the BC4F3 plant lines.

[0054] 2. Investigation of agronomic traits related to the BC4F3 plant lines

[0055] Plant the seeds of the parents Zhennuo 19, Zhennuo 20 and the introgression line BC4F3 with the RGG2-Hap6 haplotype (Zhennuo 19 RGG2 -Hap6 , Zhennuo 20 RGG2-Hap6 ) into plant lines. After maturity, take the mature seeds respectively, take pictures for observation and measurement, compare the grain morphology of rice, and further conduct statistical analysis on the 1000-grain weight and the yield per plant.

[0056] The results of Zhennuo 19 are as Figure 4 shown. There are obvious differences in grain length and grain width between Zhennuo 19 and the Zhennuo 19 introgression line with the RGG2-Hap6 haplotype (Zhennuo 19 RGG2-Hap6 ). Compared with Zhennuo 19, the grain length and grain width of Zhennuo 19 RGG2-Hap6 are significantly greater than those of Zhennuo 19. The statistical analysis results of the 1000-grain weight and the yield per plant show that, compared with Zhennuo 19, the 1000-grain weight of Zhennuo 19 RGG2-Hap6 increases by 10.07%, and the yield per plant increases by 16.95%, both showing significant differences.

[0057] The results of Zhennuo 20 are as Figure 5 shown. There are obvious differences in grain length and grain width between Zhennuo 20 and the Zhennuo 20 introgression line with the RGG2-Hap6 haplotype (Zhennuo 20 RGG2-Hap6 ). Compared with Zhennuo 20, the grain length and grain width of Zhennuo 20 RGG2-Hap6 are significantly greater than those of Zhennuo 20. The statistical analysis results of the 1000-grain weight and the yield per plant show that, compared with Zhennuo 20, the 1000-grain weight of Zhennuo 20 RGG2-Hap6 increases by 6.37%, and the yield per plant increases by 11.42%, both showing significant differences.

[0058] The above results confirm that the introduction of the RGG2-Hap6 haplotype under the backgrounds of Zhennuo 19 and Zhennuo 20 can significantly increase the grain weight and yield per plant of rice. Combining with the molecular marker primers and identification methods provided by the present invention, it is possible to determine whether the rice plants contain the RGG2-Hap6 haplotype, thereby achieving rapid, simple and efficient screening of rice genotypes. During the breeding process, the combination of molecular markers can eliminate the need for multi-year and multi-location phenotypic identification or high-cost gene analysis sequencing, reduce the breeding cost, and improve the breeding efficiency. Therefore, the present invention provides an important genetic resource for the breeding of high-yield rice varieties, and provides a breeding method for cultivating rice with increased grain weight and yield, which has good practical value and popularization value.

Claims

1. Application of rice RGG2 gene molecular marker in increasing rice grain weight and yield, characterized in that, The molecular marker of the rice RGG2 gene is the RGG2-Hap6 haplotype in the RGG2 gene haplotype. This haplotype inserts three bases GGA at the 68th base of RGG2-Hap1.

2. The application according to claim 1, wherein The amino acid sequence of the RGG2-Hap6 haplotype is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.

3.

3. A molecular marker primer for detecting the haplotype of the RGG2 gene, characterized in that, The molecular marker primers include a common forward primer RGG2-1F with a nucleotide sequence shown in SEQ ID NO.5 and two reverse downstream primers RGG2-1R and RGG2-2R with nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.7 respectively.

4. A kit, characterized in that, The kit contains the molecular marker primers described in claim 3.

5. A method for identifying the haplotype of the RGG2 gene, characterized in that, It includes the following steps: Extract the genomic DNA of the rice to be tested; using the extracted rice genomic DNA as a template, perform PCR amplification with the molecular marker primers described in claim 3.

6. The method according to claim 5, wherein If the primer pair RGG2-1F and RGG2-1R can amplify a characteristic band of 495bp, while the primer pair RGG2-1F and RGG2-2R cannot amplify a characteristic band, then the rice material carries the RGG2-Hap1 allele; if the primer pair RGG2-1F and RGG2-2R amplifies a characteristic band of 498bp, while the primer pair RGG2-1F and RGG2-1R cannot amplify a characteristic band, then the rice material carries the RGG2-Hap6 allele; if the primer pair RGG2-1F and RGG2-1R can amplify 495bp, and the primer pair RGG2-1F and RGG2-2R amplifies a characteristic band of 498bp, then the rice material carries both the RGG2-Hap1 and RGG2-Hap6 alleles.

7. The method according to claim 5, characterized in that The PCR amplification system includes molecular marker primers, 2×Taq PCR Master Mix (Novoprotein), and double-distilled water.

8. The method according to claim 5, characterized in that, The PCR amplification program is pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 30 s, for 35 cycles; extension at 72°C for 5 min.

9. A method for cultivating high-yield rice, characterized in that, Introduce RGG2-Hap6 into rice through hybridization and backcrossing until a line with significantly increased grain weight and yield per plant is obtained.

10. The method according to claim 9, wherein The primers used for identifying and screening rice plants containing the RGG2-Hap6 allele in the progeny of hybridization and backcrossing are the molecular marker primers described in claim 3.

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