Rice submergence tolerance gene LOC_Os06g17260 during germination and its application
By locating the LOC_Os06g17260 gene on rice chromosome 6 and developing molecular markers, the problem of insufficient flooding tolerance of rice seeds during the germination period was solved, the flooding tolerance and yield of rice were improved, and new genetic resources and molecular markers were provided for rice breeding.
Patent Information
- Application Number
- CN202411532975.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2025-09-19
- Estimated Expiration
- 2044-10-30
AI Technical Summary
In the prior art, insufficient research has been conducted on the submergence tolerance of rice seeds during the germination period, resulting in a low seedling emergence rate and reduced yield in direct-seeded rice fields, which limits the yield of direct-seeded rice fields.
Through GWAS analysis, the SNP site Chr: 9901755, which is significantly associated with flooding tolerance during the germination period, was located on rice chromosome 6. The LOC_Os06g17260 gene was identified, and a molecular marker based on this gene was developed. Specific primer pairs were used to amplify DNA fragments containing specific SNP sites. Different haplotypes were distinguished by sequencing, and the flooding tolerance of rice was identified.
It has improved the flooding tolerance of rice during the germination period under submerged conditions, significantly improved the flooding tolerance and yield of rice, provided genetic resources and molecular markers for rice flooding tolerance breeding, and supported the application of rice molecular breeding.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of plant genetic engineering and biotechnology, and particularly relates to a molecular marker related to a rice germination period flooding tolerance gene LOC_Os06g17260 and an application thereof. Technical Background
[0002] Rice is one of the world's most important food crops, and its production is crucial for human food security. Traditional rice cultivation methods face challenges due to labor shortages, rising costs, and climate change. Direct seeding, however, has been widely promoted for its simplicity, labor-saving, and high efficiency. However, in many rice-growing regions, particularly in direct-seeded rice systems, rice seeds are often subjected to submergence stress during germination, resulting in low seedling emergence and reduced yield. Submergence stress has become a major factor limiting direct-seeded rice yield.
[0003] Submergence tolerance in rice is a complex quantitative trait controlled by multiple genes. Studies have identified multiple genes associated with submergence tolerance in rice through methods such as QTL analysis and GWAS. However, in-depth research on the specific genes that contribute to submergence tolerance during seed germination remains insufficient. Therefore, research on genes that contribute to submergence tolerance during seed germination is of great significance and will facilitate the development of new submergence-tolerant rice varieties. Summary of the Invention
[0004] Against this background, the present invention aims to provide a novel gene for submergence tolerance during germination in rice and its associated molecular markers. To address this issue, the present invention utilized 300 japonica rice samples from a broad range of sources and rich in variation as research materials. Using GWAS, a single nucleotide polymorphism (SNP) site significantly associated with submergence tolerance during germination was identified on chromosome 6 at Chr: 9901755. Within this candidate interval, a gene associated with submergence tolerance, LOC_Os06g17260, was located at Chr6: 9996771-9998385. This gene encodes a UDP-glucosyltransferase homologous to the submergence tolerance gene OsUGT75A, which has been shown to regulate submergence tolerance during rice germination by mediating the glycosylation of abscisic acid and jasmonic acid.
[0005] Through knockout transgenic experiments, the present invention demonstrates that the LOC_Os06g17260 gene can promote rice bud elongation under submerged conditions, thereby improving submergence tolerance during the germination period. This discovery provides a solid experimental basis for the application of the LOC_Os06g17260 gene in rice breeding for submergence tolerance.
[0006] Based on three major single-base variations in the CDS region of the LOC_Os06g17260 gene, the present invention identified two major haplotypes: TAC (Hap1) and GCT (Hap2). Lines carrying Hap1 exhibited stronger submergence tolerance. Furthermore, the present invention developed a molecular marker based on this variation. This marker uses a pair of specific primers to amplify a DNA fragment containing a specific SNP site and distinguish different haplotypes through sequencing, thereby enabling the identification or auxiliary identification of submergence tolerance in rice.
[0007] Therefore, the present invention provides a rice germination period submergence tolerance gene LOC_Os06g17260, characterized in that its DNA sequence is shown in SEQ ID No. 1, or a functional equivalent or homologous gene thereof.
[0008] Specifically, there are three major single-base variations in its CDS region, namely: Chr6: 9997647, T→G (leucine→phenylalanine); Chr6: 9997559, A→C (tryptophan→glycine); Chr6: 9997514, C→T (valine→isoleucine); the gene includes two haplotypes, Hap1: TAC and Hap2: GCT.
[0009] The present invention further provides a molecular marker for identifying or assisting in identifying flooding tolerance of rice during the germination period. The molecular marker is designed based on a specific SNP variation comprising the LOC_Os06g17260 gene and is used to detect different haplotypes of the gene, among which lines carrying Hap1 exhibit stronger flooding tolerance, thereby identifying or assisting in identifying flooding tolerance of rice.
[0010] Specifically, it includes a pair of specific primers for amplifying a DNA fragment containing a specific SNP site and distinguishing different haplotypes of LOC_Os06g17260 through sequencing.
[0011] More specifically, the specific primer pair consists of the following sequences:
[0012] Primer F: single-stranded DNA molecule represented by 5′-ACGGCGTCTTGCTCCTCTC-3′;
[0013] Primer R: single-stranded DNA molecule represented by 5'-GCTCTCGCGGATGTACAGGA-3'.
[0014] The present invention thus provides a kit for identifying or assisting in identifying submergence tolerance in rice during germination, comprising a primer pair for detecting molecular markers. The molecular markers are designed based on specific SNP variations comprising the LOC_Os06g17260 gene and are used to detect different haplotypes of the gene.
[0015] Specifically, the specific primer pair consists of the following sequences:
[0016] Primer F: single-stranded DNA molecule represented by 5′-ACGGCGTCTTGCTCCTCTC-3′;
[0017] Primer R: single-stranded DNA molecule represented by 5′-GCTCTCGCGGATGTACAGGA-3′;
[0018] More preferably, primer F and primer R are mixed in a ratio of 1:1.
[0019] The present invention provides a method for identifying or assisting in identifying the flooding tolerance of rice during the germination period. The method uses the genomic DNA of the rice to be tested as a template, adopts the primer pair of the molecular marker to perform PCR amplification, and then identifies the flooding tolerance haplotype of the rice to be tested by sequencing and sequence comparison to determine the flooding tolerance of the rice during the germination period.
[0020] The present invention provides a method for cultivating or assisting in cultivating rice plants or varieties with flooding tolerance during the germination period, which comprises the steps of obtaining the flooding tolerance of a rice variety to be tested during the germination period by using the method, and selecting the rice material to be tested with a specific flooding tolerance haplotype for subsequent breeding.
[0021] The advantages of the present invention are as follows: It reveals for the first time the key role of the LOC_Os06g17260 gene in submergence tolerance during rice seed germination, providing new genetic resources and molecular markers for rice breeding for submergence tolerance. Based on non-synonymous mutations in the coding region, two major haplotypes of LOC_Os06g17260 were identified, with Hap1 exhibiting stronger submergence tolerance. Gene knockout experiments also demonstrated that LOC_Os06g17260 can improve submergence tolerance in rice under submerged conditions, providing experimental evidence for its application in rice molecular breeding. Utilizing this gene in rice breeding for submergence tolerance can significantly improve submergence tolerance and yield in direct seeding systems, with significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 : Representative images of the phenotypic variations in shoot length (a) and root length (b) among different rice accessions, scale bar = 1 cm.
[0023] Figure 2 : Frequency distribution of shoot length (a) and root length (b) of 300 rice accessions submerged in 15 cm water depth.
[0024] Figure 3 : Manhattan plot of genome-wide association analysis of shoot length (a) and root length (b) under submergence stress (p<1×10 -7 ).
[0025] Figure 4 a Sequence variation in the CDS region of LOC_Os06g17260 and the resulting haplotypes. b Local Manhattan plot (top) and LD heat map (bottom) of the 9.8 Mb to 10.0 Mb region on chromosome 6. c and d Comparison of shoot length (c) and root length (d) among different haplotypes of LOC_Os06g17260.
[0026] Figure 5 : Gene mutation types of knockout transgenic line LOC_Os06g17260 (a) and shoot length under normal (b) and submerged (c) conditions, scale bar = 1 cm. DETAILED DESCRIPTION
[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0029] Example 1. Mapping and Haplotype Analysis of the Rice Germination-Stage Submergence Tolerance Gene LOC_Os06g17260
[0030] 1. GWAS analysis of rice submergence tolerance and identification of the submergence tolerance gene LOC_Os06g17260
[0031] 1. Test materials
[0032] This study used 300 japonica rice accessions from a broad and representative natural population as the GWAS population. All seeds used in the study were oven-dried at 45°C for 7 days after harvest to break dormancy.
[0033] 2. Genotype analysis
[0034] Genotype data were processed based on resequencing information. The bcftools tool was used to filter variant sites based on the retention quality value (QUAL) greater than 20 or the INFO / DP greater than 20. The whole genome association analysis tool set called "Plink" was used to check the missing rate and remove samples with high missing rates. The minor allele frequency (MAF) > 0.05, the minimum genotype quality was 0.4 and the r 2 A total of 711,268 high-quality polymorphic SNPs were identified using a threshold greater than 0.4. SNP data were collated using RStudio Server and a Linux system, and principal component analysis (PCA) was performed. Gene annotation was completed using a Java program in Linux.
[0035] 3. Phenotypic identification of submergence tolerance during germination
[0036] Fill a clean glass with distilled water, place it in a 120℃ high-temperature sterilizer for sterilization for 20 minutes, take it out, seal it with plastic wrap and wait for the sterile water to cool completely. Select 20 full seeds of each variety and disinfect them with 1.4% sodium hypochlorite solution for 15 minutes, then wash them 5 times with sterile water, then immerse the seeds in a glass filled with sterile water with a depth of 15 cm, and culture them in a germination room (light / dark, 16h / 8h, relative humidity of 70%, constant temperature of 26℃). After 7 days of cultivation, the shoot length and root length were measured with a ruler. In this study, 10 plants of each germplasm were used to evaluate the shoot length and root length; the experiment was repeated 3 times. The results showed that the shoot length was normally distributed in the range of 0.79-4.40cm, and the root length was normally distributed in the range of 0.06-3.46cm, with coefficients of variation of 21.64% and 52.17%, respectively ( Figure 1 and Figure 2 ).
[0037] 4. GWAS analysis and candidate gene identification
[0038] A total of 711,268 high-quality SNPs were obtained from the genome sequences of 300 rice materials. GWAS analysis was performed using R and RStudioServer. A general linear model was used with a Bonferroni correction (0.05 / SNPs) and a critical p-value (P < 1 × 10 -7 ); GWAS results showed that the SNP locus located on chromosome 6 (Chr6:9901755) was co-localized in shoot length and root length (qBL6.4 and qRL6.11), and was significantly associated with submergence tolerance during seed germination, with phenotypic contribution rates of 11.12% and 10.56%, respectively ( Figure 3 By comparing the rice IRGSP-1.0 genome sequence from the MSU Rice Genome Annotation Project, version 7 (http: / / rice.plantbiology.msu.edu / ), we searched for genes related to submergence tolerance within 100 kd before and after the significant loci as candidate genes for this study.
[0039] Combined with gene annotation information, five genes homologous to OsUGT75A were located within the 9801755-10001755 region: LOC_Os06g17090, LOC_Os06g17120, LOC_Os06g17140, LOC_Os06g17220, and LOC_Os06g17260. OsUGT75A is known to regulate coleoptile length by glycosylation-mediated reduction of free ABA and JA levels, thereby modulating submergence tolerance during rice germination. Furthermore, OsUGT75A may promote leaf sheath elongation by regulating free ABA and JA levels, thereby enhancing germination of deeply sown rice seeds.
[0040] 2. Haplotype analysis of candidate genes
[0041] Based on the variation of SNPs in the coding regions of each gene, haplotypes of the five annotated genes were analyzed using RStudio Server. The significance of differences in shoot and root length between the major haplotypes, comprising more than 15 lines, was tested. The results showed that LOC_Os06g17260 has two major haplotypes: TAC (Hap1) and GCT (Hap2). There was a significant difference in shoot length between Hap1 and Hap2, while there was no significant difference in root length between the two groups. Hap1 exhibited longer shoots and was the dominant haplotype ( Figure 4 ).
[0042] Furthermore, the present invention has developed a molecular marker based on the above SNP variation. This marker uses a pair of specific primers to amplify a DNA fragment containing a specific SNP site and distinguish different haplotypes through sequencing, thereby achieving the identification or auxiliary identification of rice flooding tolerance. The primer pair is as follows:
[0043] Forward primer: 5'-ACGGCGTCTTGCTCCTCTC-3'
[0044] Reverse primer: 5′-GCTCTCGCGGATGTACAGGA-3′.
[0045] Example 2: Verification of gene knockout of LOC_Os06g17260
[0046] 1. Construction of the LOC_Os06g17260 knockout line
[0047] A knockout line (KO260) was constructed in the wild-type ZH11 background using CRISPR / Cas9 to verify gene function. The target sequence was 5'-GCCGGCGCGCCAGGCAGCCCCGG-3', and the primer sequences F: 5'-ACGATACCTCTG GCCCGAC-3', and R: 5'-GCCATACAGCCCAGACGC-3'.
[0048] 2. Phenotypic identification of submergence tolerance of KO260 during germination
[0049] 1. Screening of KO260 homozygous strains
[0050] Genomic DNA was extracted from young leaves of individual KO260 plants using the cetyltriethylammonium bromide (CTAB) method of Doyle and Dickson (1987). Primers were designed based on the target locus, with the following sequences: A: 5'-ACGATACCTCTGGCCCGAC-3'; B: 5'-GCCATACAGCCCAGACGC-3', and amplified by PCR.
[0051] The PCR amplification reaction system (20 μl) consisted of 1 μl DNA template, 1 μl (10 μM, F+R) primers, 10 μl 2× Buffer, 5 μl 2 mM dNTPs, 0.5 μl KOD FX, and 2.5 μl ddH₂O. Amplification was performed on a Professional Thermocycler (Biometra Inc.) with the following cycle: 94°C for 7 min, 98°C for 10 sec, 59°C for 30 sec, extension at 68°C for 40 sec (1 min for a 1 kb product), and 68°C for 7 min, for 34 cycles. PCR products were detected by electrophoresis on a 1.5% agarose gel (220 V constant voltage, 30 min).
[0052] If the bands were complete and clear, sequencing was performed, and sequence alignment was performed using software such as BioEdit and DNAMAN to screen out homozygous (ABI chromatogram showed a single peak) knockout transgenic plants of LOC_Os06g17260.
[0053] 2. Evaluation of flooding tolerance of KO260 during germination
[0054] The transgenic plants screened were identified for their resistance to flooding during germination according to the method in step 1 of Example 1. A control treatment group was also set up (other conditions were the same, only 25 ml of water was added). Figure 5 Under normal conditions, the shoot length of ZH11 (wild type) was 3.60 cm, and the shoot lengths of KO260 were 3.60 cm, 3.61 cm, and 3.63 cm, respectively. There was no significant difference between the shoot lengths of KO260 and ZH11. However, under submerged conditions, the shoot lengths of ZH11 were 4.02 cm, and the shoot lengths of KO260 were 3.37 cm, 3.45 cm, and 3.61 cm, respectively. There were significant differences between the shoot lengths of KO260 and ZH11 ( Figure 5 ), the shoot length of KO260 line was significantly shortened (P<0.05), indicating that LOC_Os06g17260 can promote the elongation of shoots under submerged conditions, thereby improving the submergence tolerance of rice during the germination period.
Claims
1. A method for identifying or assisting in identifying flooding tolerance of rice during the germination period, characterized in that: Using the genomic DNA of the rice to be tested as a template, PCR amplification is performed using a specific primer pair targeting the molecular marker of submergence tolerance during rice germination. Subsequently, sequencing and sequence alignment are performed to identify the submergence tolerance haplotype of the rice to be tested and determine the submergence tolerance during rice germination. The molecular marker for rice flooding tolerance during germination is a rice flooding tolerance gene during germination LOC_Os06g17260 The three single-base sites in the CDS region are Chr6:9997647, Chr6:9997559 and Chr6:9997514; If the bases of Chr6:9997647, Chr6:9997559 and Chr6:9997514 are T, A and C respectively, it indicates that the tested rice exhibits strong flooding tolerance; if the bases of Chr6:9997647, Chr6:9997559 and Chr6:9997514 are G, C and T respectively, it indicates that the tested rice exhibits weak flooding tolerance.
2. The method according to claim 1, wherein The specific primer pair consists of the following sequences: Primer F: single-stranded DNA molecule represented by 5′-ACGGCGTCTTGCTCCTCTC-3′; Primer R: single-stranded DNA molecule represented by 5'-GCTCTCGCGGATGTACAGGA-3'.
3. A method for cultivating or assisting in cultivating rice plants or varieties that are flood-tolerant during the germination period, characterized in that: The method according to claim 1 or 2 is used to select rice materials with stronger flooding tolerance for subsequent breeding.