Molecular marker for identifying excellent haplotype of rice yield gene Osicl and application thereof

Abstract

The application discloses a kind of for identifying rice yield gene OsICL Excellent haplotype OsICL HET Molecular marker and its application, the OsICL HET Haplotype is the SNP20693989 located in its gene upstream regulatory region, the CAPS primer includes the forward primer as shown in the nucleotide sequence of SEQ ID NO.1 and the reverse primer as shown in SEQ ID NO.2, for distinguishing the T or C base of physical position 20693989.The CAPS molecular marker primer pair for identifying rice kernel size developed in the application can accurately identify different haplotypes of rice kernel weight related gene OsICL Relying on the CAPS molecular marker primer pair of the application, rice kernel weight traits can be quickly and accurately identified.The method is based on DNA level detection, is not disturbed by external environment and plant growth stage, has good experimental repeatability, stable detection result, and the comprehensive performance is obviously superior to traditional field phenotype identification means.

Description

Technical Field

[0001] This invention belongs to the field of functional molecular marker technology, specifically, it relates to a method for identifying rice yield genes. OsICL DNA molecular markers of superior haplotypes and their applications. Background Technology

[0002] Rice is my country's most important food crop. Statistics show that in 2023, rice planting area accounted for approximately 24.3% of my country's total grain planting area, and its total output accounted for approximately 29.7% of my country's total grain output (data from the *China Statistical Yearbook*, 2024 edition). In recent years, affected by frequent extreme weather events and the reduction of available arable land resources, my country's food security is facing severe challenges. Therefore, exploring high-yield rice germplasm resources, analyzing the genetic basis of high-yield traits, and further increasing rice yield are of great significance for ensuring national food security and improving agricultural production efficiency.

[0003] Rice yield is primarily determined by traits such as the number of panicles per plant, the number of grains per panicle, the thousand-grain weight, and the seed setting rate. Breeding practices over the past few decades have shown that identifying and utilizing key genes regulating grain weight and the number of grains per panicle for variety improvement is an effective strategy for enhancing rice yield potential. Grain weight, as a complex quantitative trait, is influenced by multiple factors, including grain size and grain filling. The grain is an important sink organ in rice, and its size is determined by traits such as grain length, width, and thickness, making it a crucial factor affecting grain weight and yield. Grain weight is controlled by multiple quantitative trait loci (QTLs), and currently, the number of superior grain weight allelic variations available for rice variety improvement remains relatively limited. Therefore, identifying new grain weight genes with breeding application potential and their superior allelic variations is of great significance for promoting high-yield rice breeding.

[0004] OsICL The gene encodes isocitrate lyase, a key enzyme in the glyoxylate cycle. Overexpression OsICL The gene significantly increases rice grain weight and yield per plant, while knocking out the gene has the opposite effect. However, no research has yet been found on... OsICL Research reports on how differences in single nucleotide polymorphisms (SNPs) in gene regulatory regions affect gene expression levels and thus lead to differences in grain weight and yield among different rice varieties indicate that favorable haplotypes still need further investigation. Summary of the Invention

[0005] In response to the current situation OsICL To address the unresolved issue of undiscovered advantageous haplotypes in rice, this invention provides a method for identifying genes that contribute to rice yield. OsICLDNA molecular markers of superior haplotypes and their applications: Using these molecular markers, new germplasm of large-grain rice can be efficiently created through molecular marker-assisted breeding technology, and new high-yield rice varieties can be cultivated.

[0006] To achieve the above objectives, the present invention provides a method for identification. OsICL Gene OsICL HET The haplotype CAPS primer, the OsICL HET The haplotype is SNP20693989, located in the upstream regulatory region of its gene. The CAPS primers include a forward primer with nucleotide sequences as shown in SEQ ID NO. 1 and a reverse primer as shown in SEQ ID NO. 2, used to distinguish between T or C bases at physical position 20693989 (Chr7: 20693989).

[0007] The second aspect of this invention provides the above-mentioned CAPS primers for the identification of rice OsICL HET Applications in haplotypes.

[0008] The primers described above in this invention can rapidly and intuitively identify rice grain weight, making them suitable for large-scale breeding screening and shortening the breeding cycle for large-grain-weight rice. OsICL The detection of gene haplotypes can be used to screen and identify rice materials with large grain weight, thereby improving the efficiency of molecular marker-assisted selection in high-yield rice breeding.

[0009] Specifically, the identification method includes the following steps: 1) PCR amplification reaction: Using the rice genomic DNA to be tested as a template, PCR amplification was performed using the above primers, and the length of the amplification product was 333 bp. 2) Enzyme digestion and electrophoresis detection: The rice was digested with the restriction endonuclease NaeI, followed by agarose gel electrophoresis analysis. If the fragment length after digestion was still 333 bp (the amplification product was not cleaved by NaeI), it indicated that the tested rice variety carried the enzyme. OsICL HET Haplotype; if the fragment length is shortened to 164-169 bp after enzyme digestion (the amplification product is cleaved by NaeI), it indicates that the rice variety being tested carries the haplotype. OsICL LET Haplotype.

[0010] Specifically, in step 1), the PCR amplification system (20 μL) is as follows: 20 2 μL of 100 ng / μL rice genomic DNA to be tested, 10 μL of 2×Accurate Taq Master Mix (dye plus), 1 μL each of 10 μmol / L forward and reverse primers, and ddH2O to bring the total volume to 20 μL. The PCR amplification reaction conditions were as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 sec, annealing at 58℃ for 30 sec, extension at 72℃ for 25 sec, for 34 cycles; final extension at 72℃ for 5 min; and storage at 12℃.

[0011] In step 2), the enzyme digestion reaction system and reaction conditions are as follows: 10×NE Buffer 1 μL, PCR product 3 μL, NaeI restriction endonuclease 0.5 μL, and ddH2O added to make up to 10 μL; incubate at 37°C for 1 hour.

[0012] The enzyme digestion products were separated by electrophoresis on a 2% (mass fraction) agarose gel at a voltage of 120 V for 20 min. The haplotype and grain weight of the rice were determined based on the size of the enzyme digestion product bands.

[0013] A third aspect of this invention provides a method for identifying rice grain weight, which identifies which haplotype a rice variety carries. OsICL HET Haploid rice varieties have large grain weight and carry... OsICL LET Haploid rice varieties have small grain weight.

[0014] Through the above technical solution, the present invention achieves the following beneficial effects: 1. This invention develops a set of CAPS molecular marker primer pairs for identifying rice grain size, which can accurately identify rice grain weight-related genes. OsICL Different haplotypes. This detection method is simple to operate, provides intuitive observation of amplified products, and has high throughput, making it suitable for rapid screening of large-scale breeding materials.

[0015] 2. Based on the CAPS molecular marker primer pair of this invention, the grain weight trait of rice can be identified rapidly and accurately. This method is based on DNA-level detection, is not affected by external environment or plant growth stage, has good experimental repeatability, stable detection results, and its overall performance is significantly better than traditional field phenotypic identification methods.

[0016] 3. Using the marking technology of this invention to complete rice... OsICL Gene haplotype identification, combined with grain weight phenotypic data for verification, shows that the individual carries a superior haplotype. OsICL HETThe resulting rice varieties have larger grain weights. This molecular marker can be widely used in marker-assisted breeding of rice, enabling precise aggregation and targeted transfer of superior haplotypes, effectively improving grain weight traits in breeding materials, and significantly shortening the breeding cycle and increasing breeding efficiency. Attached Figure Description

[0017] Figure 1 yes OsICL Location of SNPs and Indels in gene regulatory and coding regions; Figure 2 The study analyzed the significant differences in grain weight among 387 rice varieties Hap1 to Hap7. Figure 3 yes OsICL Significant difference analysis of gene expression levels between Hap1 and Hap7; Figure 4 The dual-luciferase reporter assay clearly shows... OsICL SNP20693989 in the gene regulatory region is a superior allelic variant site; Figure 5 CAPS molecular marker detection of 20 major rice varieties OsICL Genotyping of SNP20693989 in the gene regulatory region. Detailed Implementation

[0018] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0019] Unless otherwise stated, the experimental methods in the following examples are all conventional methods, and the materials, reagents, carriers, etc. used are all commercially available.

[0020] This invention utilizes whole-genome resequencing data from 387 major rice varieties to target key regulatory genes for rice grain weight. OsICL The system identifies SNPs and Indel variant sites in the gene regulatory and coding regions. Based on the genotyping results of these variant sites, the... OsICL The genes were precisely classified into seven haplotypes, Hap1 to Hap7. Further integration of phenotypic analysis of grain weight, gene expression level, and promoter enzyme activity verification results from different haplotype materials systematically clarified the... OsICL The invention identifies specific functional sites corresponding to superior allelic variations in genes. Based on these specific site characteristics, the invention specifically develops CAPS molecular markers for high-throughput and rapid identification of rice grain weight traits. The specific implementation of this invention is as follows: Example 1 OsICL Haplotype analysis of genes Based on whole-genome resequencing data from 387 major rice varieties, targeting rice OsICL Variant site mining and sequence feature analysis were conducted on the regulatory and coding regions of genes. Through systematic alignment, screening, and validation, the final results were... OsICL A total of 20 single nucleotide variant sites (SNPs) and 2 insertion / deletion variant sites (InDel) were identified within the gene segment. For details on the distribution and characteristics of these variant sites, please refer to [link to relevant documentation]. Figure 1 .

[0021] Based on the combined characteristics of all SNPs and InDel variant sites obtained from the above identification, haplotype typing analysis was performed on 387 tested rice varieties, and all tested materials were accurately classified into 7 natural haplotypes (Hap1 ~ Hap7).

[0022] Example 2 (Different) OsICL Analysis of Grain Weight Phenotypic Differences in Haplotype Rice Varieties To clarify OsICL To investigate the association between different haplotypes of the gene and the grain weight trait of rice, the above 387 major rice varieties were uniformly cultivated and managed. At the maturity period, the phenotypic indicators related to grain weight of each variety were measured, and the differences in grain weight phenotypes of different haplotype materials were compared and analyzed.

[0023] Phenotypic statistical results are as follows Figure 2 As shown: in 7 types OsICL Among the haplotypes, rice varieties carrying haplotypes Hap1 and Hap2 had significantly higher grain weights than rice materials corresponding to haplotypes Hap3, Hap4, Hap5, Hap6, and Hap7, with statistically significant differences between groups. This result confirms that... OsICL The Hap1 and Hap2 haplotypes are excellent natural haplotypes that regulate rice grain weight increase and can serve as excellent genotype targets for rice large-grain germplasm screening and molecular marker-assisted breeding.

[0024] Example 3: Rice materials with different haplotypes OsICL Validation of differences in gene expression levels To further reveal the differences OsICL This study investigates the molecular mechanisms by which haplotypes regulate grain weight in rice and elucidates the correlation between haplotype variation and gene expression levels. Representative rice materials corresponding to the seven haplotypes (Hap1–Hap7) were selected for further investigation. OsICL Gene relative expression level detection and analysis.

[0025] The real-time quantitative PCR (qRT-PCR) technique was used to determine the content of each material. OsICL Gene transcriptional expression levels, in rice OsActin The gene was used as an internal reference gene, using 2-ΔΔCt Method calculation OsICL The relative expression level of genes ensures the accuracy and reliability of the test results.

[0026] According to rice OsICL Specific qRT-PCR amplification primers were designed based on the conserved full-length cDNA sequence of the gene. The upstream primer is SEQ ID No. 3, and the downstream primer is SEQ ID No. 4. Simultaneously, according to... OsActin Internal reference primers were designed based on conserved gene sequences. The upstream primer is SEQ ID No. 5, and the downstream primer is SEQ ID No. 6. The specific primer sequences are as follows: SEQ ID No.3: 5'-TCTACGACAGGGTGCTCCAAGAC-3'; SEQ ID No.4: 5'-TGCTCTTCAGTAACTCCTTTGCC-3'; SEQ ID No.5: 5'-CCCCTCCTGAAAGGAAGTA-3'; SEQ ID No. 6: 5'-GGTCCGAAGAATTAGAAGCA-3'.

[0027] Gene expression level detection results as follows Figure 3 As shown: Hap1 and Hap2 haplotype rice materials OsICL The relative expression level of the gene was significantly higher than that of the five haplotypes Hap3–Hap7. This, combined with the phenotypic results of Example 2, confirms that... OsICL The high expression level of the gene is an important molecular basis for the increase in rice grain weight. At the same time, it further verifies that Hap1 and Hap2 are excellent haplotypes that regulate the large grain trait in rice, providing a theoretical basis and technical support for carrying out high-yield molecular breeding of rice using this gene and haplotype.

[0028] Example 4: Screening using a dual-luciferase reporter assay OsICL Key functional variation sites of genes Based on the aforementioned haplotype classification and gene expression results, the superior haplotype and the non-superior haplotype were analyzed. OsICL Multiple sequence alignment analysis of gene promoter sequences revealed that the superior haplotypes Hap1 and Hap2 differed from the other five non-superior haplotypes in... OsICL There are two stable shared SNP variant sites in the gene promoter region, named SNP-822 and SNP-543, respectively. For detailed information on the variant sites and their locations, please refer to [link to relevant documentation]. Figure 1 To accurately screen and identify regulatory measures OsICL The core functional variation sites that influence gene expression and thus affect grain weight trait in rice were used to construct different types of [data / samples]. OsICLThe dual-luciferase activity of the gene promoter recombinant vector was verified.

[0029] The superior haplotype promoter sequence (HET) and the inferior haplotype promoter sequence (LET) were cloned, and corresponding single-point mutation vectors (M1 and M2) were constructed targeting the two SNP sites. All promoter fragments were ligated into pGreen-0800 dual-luciferase reporter vectors to obtain recombinant expression vectors. The constructed recombinant vectors were transformed into rice protoplasts, and the differences in transcriptional activity between different promoter fragments were analyzed by detecting the relative activities of firefly luciferase (LUC) and Renilla luciferase (REN) in each group. The specific amplification primer sequences used for vector construction in this example are as follows: SEQ ID No. 7: 5'-CCCCCTCGAGGTCGACATGTATCTGCATATAGTTAA-3'; SEQ ID No. 8: 5'-TTGGCGTCTCCATGGGATAACCAAGAGTACACTGC-3'.

[0030] Dual-luciferase activity assay results are as follows Figure 4 As shown: Compared with the transcriptional activity of the low-expression type LET promoter, the promoter fragment with M2 site-directed mutation at SNP-543 (physical location: Chr7: 20693989) showed a significant increase in transcriptional activity, and the activity level was not significantly different from that of the high-expression type HET excellent promoter; while the activity of the promoter with M1 point mutation at SNP-822 did not show a significant increase, and was basically consistent with the level of low-activity LET promoter.

[0031] The above experimental results confirm that SNP-543, located at Chr7: 20693989, is the cause of... OsICL The core functional variation site that differentiates the expression levels of different allelic isoforms of a gene. Based on the genotypic differences at this key variation site, rice... OsICL The gene haplotypes were reclassified into two categories: one category is the superior type with high gene expression and corresponding large grain weight trait ( OsICL HET Another type is the common type with low gene expression, corresponding to the small particle weight trait ( OsICL LET ).

[0032] Example 5: Development of CAPS molecular markers targeting key functional sites in rice grain weight trait To achieve OsICLRapid and accurate molecular identification of superior haplotypes serves the screening of rice large-grain-weight germplasm resources and molecular marker-assisted breeding. In this embodiment, specific typing primers were designed using CAPS marker development technology for the core functional variant site SNP-543 (physical location: Chr7:20693989).

[0033] Using the conserved sequences upstream and downstream of this SNP site as templates, a pair of specific CAPS genotyping primers were designed, with corresponding sequences SEQ ID NO.1 and SEQ ID NO.2. Carrying OsICL LET The haplotype rice genomic DNA amplification product contains a NaeI restriction endonuclease recognition site (GCCGGC), while OsICL HET The haplotype amplification product does not have this restriction enzyme site, so the two haplotypes can be accurately classified by the difference in restriction enzyme digestion.

[0034] SEQ ID NO.1: 5'-ACGTTGTAGCCATCAAAACG-3'; SEQ ID NO. 2: 5'-CGGGATTTTGATCGGTTTCG-3'.

[0035] Example 6: Verification of the accuracy and reliability of CAPS molecular markers To verify the genotyping accuracy and practicality of the CAPS molecular markers developed in Example 5, 20 representative major rice varieties were randomly selected as experimental materials. Genomic DNA was extracted from each sample, and specific amplification primer pairs SEQ ID NO.1 and SEQ ID NO.2 were used for... OsICL PCR amplification was performed on the target region of the gene promoter.

[0036] All PCR amplification products were sequenced for verification and sequence alignment analysis. Genotyping was performed based on the base type at SNP-543 (Chr7:20693989): test materials with the base T at this site were identified as... OsICL HET Superior haplotype, corresponding to the large-grain-weight phenotype in rice; test materials with a C base at this site were identified as... OsICL LET Non-superior haplotypes correspond to the small grain weight phenotype in rice. The specific genotyping results for the 20 materials are detailed in Table 1.

[0037] Simultaneously, the CAPS-specific primer pairs (SEQ ID NO.1, SEQ ID NO.2) developed in Example 5 were used to perform batch PCR amplification of the genomic DNA of the above 20 rice materials, obtaining specific amplification products. Subsequently, all PCR products were digested with NaeI restriction endonuclease at an isothermal temperature. The fragment size of the digested products was detected by agarose gel electrophoresis, and genotyping was performed based on the electrophoretic bands. The specific determination criteria are as follows: If the electrophoretic band length of the enzyme digestion product is 333 bp, it indicates that the PCR amplification product was not cleaved by the NaeI restriction enzyme, and the rice material being tested carries the enzyme. OsICL HET Haplotype, characterized by large grain weight; if a 164-169 bp band is produced after enzyme digestion, it indicates that the amplification product has been specifically cleaved by NaeI restriction enzyme, and the tested rice material carries the [specific trait]. OsICL LET It is a non-superior haplotype, exhibiting small grain weight characteristics.

[0038] Batch typing test results are as follows Figure 5 As shown, the genotyping results of 20 rice materials using the CAPS molecular marker developed in this invention are completely consistent with the gene sequencing genotyping results, with no mismatches or misclassifications. In summary, this demonstrates that the CAPS molecular marker targeting the SNP-543 functional site developed in this embodiment has high specificity and accuracy, and can rapidly and efficiently achieve genotyping of rice... OsICL The batch identification of haplotypes with superior grain weight is applicable to scenarios such as screening of large-grain rice germplasm, molecular-assisted breeding, and rapid genotype detection, and has extremely high reliability and field application value.

[0039] The PCR amplification system (20 μL) is as follows: 20 2 μL of 100 ng / μL rice genomic DNA to be tested, 10 μL of 2×Accurate Taq Master Mix (dye plus), 1 μL each of 10 μmol / L forward and reverse primers, and ddH2O to bring the total volume to 20 μL. The PCR amplification reaction conditions were as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 sec, annealing at 58℃ for 30 sec, extension at 72℃ for 25 sec, for 34 cycles; final extension at 72℃ for 5 min; and storage at 12℃.

[0040] The enzyme digestion reaction system and conditions were as follows: 1 μL of 10×NE Buffer, 3 μL of PCR product, 0.5 μL of NaeI restriction endonuclease, and ddH2O to a final volume of 10 μL. Incubate at 37°C for 1 hour.

[0041] The enzyme digestion products were separated by electrophoresis on a 2% (mass fraction) agarose gel at a voltage of 120 V for 20 min. The haplotype and grain weight of the rice were determined based on the size of the enzyme digestion product bands.

[0042] Table 1. 20 Main Rice Varieties OsICL Haplotype of a gene

[0043] In summary, this invention targets key regulatory genes for rice grain weight. OsICL Systematic studies on natural variation mining, haplotype typing, and functional verification were conducted. This invention analyzes rice... OsICL Sequence variation characteristics of gene regulatory and coding regions were analyzed, and several SNPs and InDel variation sites were identified. Based on the variation combination characteristics, the natural rice population was divided into 7 species, Hap1 to Hap7. OsICL Gene haplotypes. Population phenotypic identification confirmed that Hap1 and Hap2 are superior haplotypes that regulate rice grain weight and can significantly increase rice grain weight.

[0044] This invention further combines differential gene expression analysis with functional verification using a dual-luciferase reporter system to precisely pinpoint the location of gene expression on chromosome 7 of rice. OsICL The Chr7:20693989 (SNP-543) site in the upstream regulatory region of the gene is the determining factor. OsICL The core functional variation site that regulates gene expression differences and thus the grain weight trait in rice. Based on this key functional site, this invention specifically developed a set of efficient and precise CAPS molecular markers, which can achieve precise genotyping at the SNP-543 site and effectively differentiate rice varieties. OsICL Superior haplotypes of genes ( OsICL HET ) and non-superior haplotypes ( OsICL LET ).

[0045] Batch testing and sequencing verification of multiple major rice varieties confirmed that the molecular marker typing results were accurate and reproducible, and the selected samples were effective. OsICL HET Superior haplotype rice materials exhibit significantly better grain weight characteristics than non-superior haplotype materials. Therefore, the molecular markers developed in this invention can be efficiently applied to rice. OsICL HET Rapid identification of superior haplotypes, screening of large-grain germplasm resources, and molecular marker-assisted high-yield breeding can effectively overcome the technical bottlenecks of low accuracy and long cycle in traditional phenotypic breeding, significantly improve the selection efficiency and breeding accuracy of high-yield and high-quality rice, and have good scientific research value and broad breeding application prospects.

[0046] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0047] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0048] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for identification OsICL Gene OsICL HET The haplotype CAPS primer is characterized by, The OsICL HET The haplotype is SNP20693989, located in the upstream regulatory region of its gene. The CAPS primers include a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer as shown in SEQ ID NO.2, used to distinguish between T or C bases at physical position 20693989.

2. The CAPS primers as described in claim 1 for identifying rice OsICL HET Applications in haplotypes.

3. The application according to claim 2, characterized in that, The identification method includes the following steps: 1) Using the rice genomic DNA to be tested as a template, PCR amplification was performed using the primers described above, and the amplification product was 333 bp in length; 2) The rice was digested with the restriction endonuclease NaeI, followed by agarose gel electrophoresis analysis. If the fragment length after digestion was still 333 bp, it indicated that the tested rice variety carried the virus. OsICL HET Haplotype; if the fragment length is shortened to 164-169 bp after enzyme digestion, it indicates that the rice variety being tested carries the haplotype. OsICL LET Haplotype.

4. The application according to claim 3, characterized in that, In step 1), the PCR amplification system is as follows: 20 2 μL of 100 ng / μL rice genomic DNA to be tested, 10 μL of 2×Accurate Taq Master Mix, 1 μL each of 10 μmol / L forward and reverse primers, and ddH2O to bring the total volume to 20 μL; The PCR amplification reaction conditions were as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 sec, annealing at 58℃ for 30 sec, extension at 72℃ for 25 sec, for 34 cycles; final extension at 72℃ for 5 min; and storage at 12℃.

5. The application according to claim 3, characterized in that, In step 2), the enzyme digestion reaction system and reaction conditions are as follows: 10×NE Buffer 1 μL, PCR product 3 μL, NaeI restriction endonuclease 0.5 μL, and ddH2O added to make up to 10 μL; incubate at 37°C for 1 hour.

6. The application according to claim 3, characterized in that, In step 2), the enzyme digestion products are separated by electrophoresis on a 2% agarose gel at a voltage of 120 V for 20 min.

7. A method for identifying the size of rice grains, characterized in that, carry OsICL HET Haploid rice varieties have large grain weight and carry... OsICL LET Haploid rice varieties have small grain weight.

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