Method for analyzing dominant allelotype of stalk strength gene based on MBKbase database

Abstract

The invention belongs to the field of rice molecular genetics, and particularly discloses an MBKbase database-based dominant allelotype analysis method for stem strength genes, which comprises the following steps: firstly, analyzing different genotypes of rice stem strength genes SCM3 and OsRLCK191, and according to different genotype germplasm resources of the rice stem strength genes carried in MBKbase-Rice, constructing a dominant allelotype of the rice stem strength genes SCM3 and OsRLCK191; the method comprises the following steps: selecting rice materials with single-dominant allelic genes, double-dominant allelic genes and non-dominant allelic genes (reference genome type), determining the stem strength character of rice germplasm resources of different genotype combinations of the rice stem strength gene, and finally performing sequencing verification on different genotypes of the rice stem strength gene. Finally, clearly breeding a strategy that the stalks are thick and strong, the stalks are high in elasticity or the stalks are moderate in thickness and elasticity to improve the lodging resistance of the rice variety; therefore, low-cost and high-throughput genotype screening can be realized.

Description

Technical Field

[0001] This invention relates to the field of rice molecular genetics, and in particular to a method for analyzing the dominant allele genotypes of stem strength genes based on the MBKbase database. Background Technology

[0002] Lodging is one of the main factors restricting high yield and quality in rice. Once lodging occurs, the deterioration of the canopy structure leads to a decrease in photosynthetic rate, which in turn inhibits the translocation of nutrients and water; rice grains exposed to high humidity for a long time will sprout in the panicle, resulting in poor grain quality. Globally, lodging causes a 12%-25% reduction in rice yield annually (FAO, 2024). In the main japonica rice producing areas of Northeast China, lodging caused by strong winds and rain during the grain-filling stage can reduce the thousand-grain weight by 18.3% and increase the chalky grain rate by 22.7%. Traditional lodging-resistant breeding relies on phenotypic selection, but stem strength, as a quantitative trait, is regulated by multiple genes with minor effects, resulting in low selection efficiency (breeding cycles as long as 8-10 years). With the increasing frequency of extreme weather events and the widespread use of mechanized harvesting, breeding new varieties that combine lodging resistance with high quality and yield has become a strategic need to ensure food security.

[0003] Breeding lodging-resistant rice varieties is a crucial measure for ensuring food security. During the "Green Revolution," the high density tolerance and lodging resistance of dwarf varieties led to a significant increase in overall yield. However, their strong fertilizer tolerance resulted in low nitrogen absorption and utilization efficiency, and the excessive application of nitrogen fertilizer, while increasing yield, also damaged the soil and environment. In the post-Green Revolution era, breeding super rice often focuses on increasing biomass, and appropriately increasing rice plant height is one of the key ways to improve biomass yield. However, this also places higher demands on the stem strength of these varieties. Therefore, the utilization of stem strength-related genes plays an important role in improving the efficiency of lodging-resistant breeding in japonica rice. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for analyzing the dominant allele genotypes of stem strength genes based on the MBKbase database, thereby achieving low-cost, high-throughput genotype screening.

[0005] To achieve the above objectives, the present invention is implemented according to the following technical solution: The method for analyzing the dominant alleles of stem strength genes based on the MBKbase database includes the following steps: S1. Analyze the different genotypes of rice stem strength genes based on the MBKbase-Rice database; S2. Based on the dominant allele genotypes of rice stem strength genes in MBKbase-Rice, select rice materials with single dominant alleles, double dominant alleles, and reference genotypes. S3. Determine the stem strength traits of rice germplasm resources with different genotype combinations of the rice stem strength gene. The stem strength traits include section coefficient, bending moment, breaking moment, bending stress, and lodging index. S4. Sequencing verification of different genotypes of the rice stem strength gene: During the tillering stage, leaves from 12 rice varieties were selected, and DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method; 5 μl of DNA solution was taken and electrophoresed with 1% agarose and 1XTAE buffer at 120-180 V; the concentration and purity were detected by spectrophotometer; the whole segment of the rice stem strength gene was amplified using the whole genome DNA of each variety as a template, and the rice stem strength gene was subjected to Sanger sequencing to identify the distribution of different genotypes of the rice stem strength gene in the 12 rice varieties, in order to verify the genotype analysis of the MBKbase database.

[0006] Preferably, the rice stem strength gene is a rice... SCM3 Genes and rice OSRLCK191 Gene.

[0007] Further, step S1 specifically includes: accessing the MBKbase database, entering the search terms "SCM3" and "RLCK191" under the "KnownGene" menu, and extracting whole-genome SNP data from 5140 Chinese rice germplasms; filtering criteria: retaining SNP sites with a minimum allele frequency (MAF) ≥ 0.05 and a deletion rate < 10%.

[0008] Furthermore, step S3 specifically includes: Select SCM3 and O S RLCK191 Cold-region japonica rice germplasm resources with different genotype combinations were sampled approximately 15 days after heading to analyze the corresponding stem strength traits. Stem strength traits were measured using a YYD-1 stem strength measuring instrument, and the calculation method is as follows: Section coefficient SM=π (a2) 3 a1-b2 3 b1) / 32a2, where a2 and a1 represent the short and long outer diameters of the stem, respectively, and b2 and b1 represent the short and long inner diameters of the stem, respectively; Bending moment WP = distance from the internode to the top of the ear × fresh weight from the break point of the internode to the top of the ear; Breaking moment = M (maximum tensile force at stem break × distance between fulcrums) / 4; Bending stress BS = M / SM; The lodging index LI = WP / M.

[0009] Compared with existing technologies, this invention first analyzes different genotypes of rice stem strength genes. Based on the dominant allele genotypes of rice stem strength genes in MBKbase-Rice, it then selects rice materials with single dominant alleles, double dominant alleles, and reference genotypes to determine the stem strength trait of rice germplasm resources with different genotypes of the rice stem strength gene. Finally, it verifies the different genotypes of rice stem strength genes through sequencing. Ultimately, it clarifies the strategy of selecting varieties with robust stems, high stem elasticity, or moderate stem thickness and elasticity to improve the lodging resistance of rice varieties. It is evident that this invention can achieve low-cost, high-throughput genotype screening. Attached Figure Description

[0010] Figure 1 For MBKbase database SCM3 Stem diameter of materials with different allelic genotypes.

[0011] Figure 2 For MBKbase database OsRLCK191 Stem diameter of materials with different allelic genotypes.

[0012] Figure 3 for SCM3 and OsRLCK191 Stem strength traits of the second and third internodes from the bottom in different allelic combinations: (A) bending moment; (B) breaking moment; (C) bending stress; (D) section coefficient; (E) lodging coefficient.

[0013] Figure 4 for SCM3 and OsRLCK191 Relationship between section coefficient and bending stress in different allelic genotype combinations (Note: the curve in the figure represents the breaking moment): (A) Rice stem second internode from the bottom; (B) Rice stem third internode from the bottom.

[0014] Figure 5 for SCM3 Two genotypes were sequenced.

[0015] Figure 6 for OsRLCK191 Two genotypes were sequenced. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0017] Example 1: Based on the MBKbase database SCM3 and OsRLCK191 Different genotype analysis In recent years, large rice databases containing sequencing and phenotypic data have provided valuable materials and knowledge for rice research and breeding projects. MBKbase-Rice is an integrated omics database that includes rice germplasm information, multiple reference genomes with a unified locus, population sequencing data, phenotypic data, known alleles, and gene expression data. Using this database, we analyzed previously reported genes controlling lodging resistance in rice and, combined with relevant literature reports, ultimately identified… SCM3 and OsRLCK191 Dominant alleles: SCM3 The dominant alleles were the same as GID3 (Table 1), while approximately 70% of the materials with the GID3 genotype had stem diameters ≥5 mm. Figure 1 Furthermore, GID3 contains a total of 51 cold-region japonica rice materials, such as Longjing 31 and Songjing 9. OsRLCK191 The dominant alleles were the same as those of GID3 (Table 2). OsRLCK191 Approximately 60% of the materials with genotype GID3 have a stem diameter ≥5mm. Figure 2 (Among them, there are about 57 samples of Northeast japonica rice.)

[0018] Table 1. MBKbase-Rice Database SCM3 Allele analysis

[0019] Note: In the Ref line, the letter N represents multiple consecutive bases, indicating that the variation at that site in the corresponding germplasm resource is a deletion. In the GID line, uppercase letters indicate that the variation at that site is homozygous, "-" indicates a read deletion, and "0" indicates that it is identical to the reference sequence.

[0020] Table 2. MBKbase-Rice Database OsRLCK191 Allelic analysis

[0021] Example 2: SCM3 and OsRLCK191 Materials with different dominant allele combinations According to MBKbase-Rice SCM3 and OsRLCK191 The dominant allele germplasm resources were selected from the main japonica rice varieties cultivated in Northeast China that possess single dominant alleles, double dominant alleles, and reference genotypes, as shown in Table 3.

[0022] Table 3 SCM3 and OsRLCK191 Dominant allele combination materials

[0023] Example 3: SCM3 and OsRLCK191 Stalk strength identification of different genotypes of japonica rice varieties According to the results of the analysis of variance (Table 4), SCM3 Allelic type affects the bending moment (p<0.001) and lodging index (p<0.05) of the second internode from the bottom, and affects the bending moment (p<0.001), cross section coefficient (p<0.05), and lodging index (p<0.010) of the third internode from the bottom. OsRLCK191 Allelic genotype affects other mechanical traits besides breaking moment, such as bending moment between the second and third segments from the bottom (p<0.001), section modulus (p<0.001), bending stress (p<0.001), and lodging index (p<0.05). SCM3 and OsRLCK191 Allelic interactions primarily affect the bending moment of the second internode from the bottom (p<0.05) and the lodging index of the third internode from the bottom (p<0.05).

[0024] Table 4. Tests for inter-subject effects

[0025] Depend on Figure 3 middle SCM3 and OsRLCK191 The mechanical characteristics of different alleles show that, with SCM3 -GID2 / OsRLCK191 Compared to -GID2, SCM3 -GID3 / OsRLCK191 -GID2 has no significant effect on the mechanical characteristics between the second and third sections from the bottom; SCM3- GID2 / OsRLCK191- The GID3 combination significantly reduces the bending moment, section modulus, and collapse index between the second and third sections from the bottom, while significantly increasing the bending stress. SCM3 -GID2 / OsRLCK191 Compared to -GID3, SCM3 -GID3 / OsRLCK191 -GID3 combination significantly improves the bending moment and lodging index between the second and third segments from the bottom; with SCM3 -GID3 / OsRLCK191 Compared to -GID2, SCM3 -GID3 / OsRLCK191 The -GID3 combination has no significant effect on the mechanical characteristics between the second and third sections from the bottom. The above results indicate that... SCM3 and OsRLCK191 The regulation of stem strength depends on the allele type of the other.

[0026] The relationship between section coefficient and bending stress (Figure 4) shows that bending stress decreases with increasing section coefficient, meaning that thicker stems have lower elasticity. The breaking moment is affected by both section coefficient and bending stress. Longjing 38, with its large section coefficient, maintains a relatively large breaking moment; Hejiang 23, with its small section coefficient, maintains a high breaking moment due to high bending stress; Suijing 18, with its moderate section coefficient and bending stress, also maintains a relatively high breaking moment. These results also reflect that three main strategies were adopted in the breeding of lodging-resistant japonica rice in cold regions: selecting varieties with robust stems, high stem elasticity, or moderate stem thickness and elasticity to improve lodging resistance.

[0027] Example 4: SCM3 and OsRLCK191 Sequencing verification of different genotypes SCM3 As a transcription factor, its genotype variation was found in the 5' UTR region through database analysis. Therefore, the amplified region includes the 5' UTR and CDS regions, totaling 1672bp. Thus, two pairs of primers were designed to cover this gene region. OsRLCK191 This gene is a cytoplasmic receptor kinase, containing exons and introns totaling 2354 bp in the genome. Therefore, five pairs of primers were designed to cover this gene region. Primers were designed for segmental amplification of the two genes (Table 5), and the amplified PCR products were Sanger sequenced and assembled.

[0028] Sequencing results show that SCM3 The gene has a 4bp GTGT insertion at position +230bp in the 5'UTR region. This genotype is carried by Dongnong 425, Suijing 18, Longjing 31, Songjing 9, and Longjing 38, and is identified as the GID3 genotype by MBKbase. Conversely, the genotype without the insertion is the GID2 type. Figure 5 ). OsRLCK191 The gene shows a C / T change at the SNP site at +83 bp in exon 2; a G / C change at the SNP site at +228 bp in exon 4; and a T / G change at the SNP site at +431 bp in exon 4. Figure 6 ).

[0029] Table 5 SCM3 and OsRLCK191 Gene sequencing primers

[0030] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for analyzing dominant alleles of stem strength genes based on the MBKbase database, characterized in that, Includes the following steps: S1. Analyze the different genotypes of rice stem strength genes based on the MBKbase-Rice database; S2. Based on the dominant allele genotypes of rice stem strength genes in MBKbase-Rice, select rice materials with single dominant alleles, double dominant alleles, and reference genotypes. S3. Determine the stem strength traits of rice germplasm resources with different genotype combinations of the rice stem strength gene. The stem strength traits include section coefficient, bending moment, breaking moment, bending stress, and lodging index. S4. Sequencing verification of different genotypes of the rice stem strength gene: During the tillering stage, leaves from 12 rice varieties were selected, and DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. 5 μl of DNA solution was mixed with 1% agarose and 1X TAE buffer solution and subjected to electrophoresis at 120–180 V. The concentration and purity were measured using a spectrophotometer. The entire segment of the rice stem strength gene was amplified using the whole-genome DNA of each variety as a template, and Sanger sequencing was performed on the rice stem strength gene to identify the distribution of different genotypes of the rice stem strength gene in the 12 rice varieties, thus verifying the genotype analysis capabilities of the MBKbase database.

2. The method for analyzing the dominant allele genotypes of stem strength genes based on the MBKbase database according to claim 1, characterized in that, The rice stem strength gene is rice SCM3 Genes and rice OsRLCK191 Gene.

3. The method for analyzing the dominant allele genotypes of stem strength genes based on the MBKbase database according to claim 2, characterized in that, Step S1 specifically includes: accessing the MBKbase database, entering the search terms "SCM3" and "RLCK191" under the "KnownGene" menu, and extracting whole-genome SNP data from 5140 Chinese rice germplasms; filtering criteria: retaining SNP sites with a minimum allele frequency (MAF) ≥ 0.05 and a deletion rate < 10%.

4. The method for analyzing the dominant allele genotypes of stem strength genes based on the MBKbase database according to claim 3, characterized in that, Step S3 specifically includes: Select SCM3 and OSRLCK191 Cold-region japonica rice germplasm resources with different genotype combinations were sampled approximately 15 days after heading to analyze the corresponding stem strength traits. Stem strength traits were measured using a YYD-1 stem strength measuring instrument, and the calculation method is as follows: Section coefficient SM=π (a2) 3 a1-b2 3 b1) / 32a2, where a2 and a1 represent the short and long outer diameters of the stem, respectively, and b2 and b1 represent the short and long inner diameters of the stem, respectively; Bending moment WP = distance from the internode to the top of the ear × fresh weight from the break point of the internode to the top of the ear; Breaking moment = M (maximum tensile force at stem break × distance between fulcrums) / 4; Bending stress BS = M / SM; The lodging index LI = WP / M.

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