Rice germination period flooding resistance gene LOCOs06g17260 and application thereof

Abstract

The invention discloses a paddy rice germination period flood tolerance gene LOCOs06g17260 for coding UDP-glucosyltransferase and application of the paddy rice germination period flood tolerance gene LOCOs06g17260 in paddy rice germination period flood tolerance character identification and breeding. According to the invention, GWAS analysis is utilized, an SNP site which is significantly related to the flooding resistance of rice in the germination period is associated to a chromosome 6, a flooding resistance related gene LOCOs06g17260 is identified in a candidate interval of the chromosome 6, and the bud length difference of different haplotypes of the gene under a flooding condition is significant. A molecular marker for detecting gene functional SNP variation is provided, the marker amplifies a DNA fragment containing a specific SNP site through a pair of specific primers, and different haplotypes are distinguished through sequencing. A gene knockout experiment proves that the LOCOs06g17260 can promote the elongation of buds under a submerging condition and improve the flooding resistance of rice in a germination period, and has a wide application prospect.

Description

Technical Field

[0001] The 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 most important food crops in the world, and its production is vital to human food security. Traditional rice cultivation methods face challenges due to labor shortages, rising costs, and climate change, while direct seeding has been widely promoted because of its simplicity, labor-saving, and high efficiency. However, in many rice-growing areas, especially in direct-seeded rice systems, rice seeds are often subjected to submergence stress during the germination period, resulting in low seedling emergence rate and reduced yield. Submergence stress has become one of the important factors limiting the yield of direct-seeded rice.

[0003] Rice flooding tolerance is a complex quantitative trait controlled by multiple genes. At present, studies have identified multiple genes related to flooding tolerance in rice through QTL analysis and GWAS. However, in-depth research on specific genes for flooding tolerance during seed germination is still insufficient. Therefore, research on flooding tolerance genes during seed germination is of great significance and is conducive to the development of new flood-tolerant rice varieties. Summary of the invention

[0004] In view of the above research background, the present invention aims to provide a new gene for flooding tolerance during the germination period of rice and its related molecular markers. In order to solve this problem, the present invention uses 300 japonica rice with a wide range of sources and rich variations as research materials, and uses GWAS to associate a SNP site on chromosome 6 that is significantly associated with flooding tolerance during the germination period, the position is Chr: 9901755, and locates the gene LOC_Os06g17260 related to flooding tolerance in its candidate interval, and its specific position is Chr6: 9996771-9998385. This gene encodes UDP-glucosyltransferase, which is homologous to the flooding tolerance gene OsUGT75A. OsUGT75A has been proven to regulate the flooding tolerance of rice during the germination period by mediating the glycosylation process of abscisic acid and jasmonic acid.

[0005] Through knockout transgenic experiments, the present invention proves that the LOC_Os06g17260 gene can promote the elongation of rice buds under submerged conditions, thereby improving the submergence tolerance of rice during the germination period. This discovery provides a solid experimental basis for the application of the LOC_Os06g17260 gene in rice submergence tolerance breeding.

[0006] Based on the 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), among which the strain carrying Hap1 showed stronger flooding tolerance. Furthermore, the present invention developed a molecular marker based on the variation, which amplifies a DNA fragment containing a specific SNP site through a pair of specific primers and distinguishes different haplotypes through sequencing, thereby realizing the identification or auxiliary identification of rice flooding tolerance.

[0007] Therefore, the present invention provides a rice germination period flooding tolerance gene LOC_Os06g17260, characterized in that its DNA sequence is as shown in SEQ ID No.1, or its functional equivalent or homologous gene.

[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, Hap2: GCT.

[0009] The present invention further provides a molecular marker for identifying or assisting in identifying the 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, wherein the strain carrying Hap1 exhibits stronger flooding tolerance, so as to identify or assist in identifying the 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 flooding tolerance of rice during germination, which comprises a primer pair for detecting molecular markers, wherein 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 invention provides a method for identifying or assisting in identifying flooding tolerance of rice during germination period, which uses genomic DNA of 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 rice during germination period.

[0020] The present invention provides a method for cultivating or assisting in cultivating rice plants or varieties with flood tolerance during the germination period, which comprises the steps of obtaining the flood tolerance of the rice variety to be tested during the germination period by adopting the method, and selecting the rice material to be tested with a specific flood tolerance haplotype for subsequent breeding.

[0021] The advantages of the present invention are: the present invention reveals for the first time the key role of the LOC_Os06g17260 gene in the flooding tolerance of rice seeds during the germination period, and provides new gene resources and molecular markers for rice flooding tolerance breeding. Based on non-synonymous mutations in the coding region, two main haplotypes of LOC_Os06g17260 were identified, and Hap1 showed strong flooding tolerance. Gene knockout experiments also proved that LOC_Os06g17260 can improve the flooding tolerance of rice under flooding conditions, providing an experimental basis for its application in rice molecular breeding. Using this gene for rice flooding tolerance breeding can significantly improve the flooding tolerance and yield of rice in a direct seeding system, which has important economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Representative diagrams of the phenotypic variation of shoot length (a) and root length (b) in different rice accessions, Scale bar = 1 cm.

[0023] Figure 2 : Frequency distribution of shoot length (a) and root length (b) of 300 rice materials 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 (upper) and LD heat map (lower) of the 9.8Mb to 10.0Mb region of chromosome 6. c and d Comparison of shoot length (c) and root length (d) differences 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 flooding (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. Location and haplotype analysis of the rice germination flooding tolerance gene LOC_Os06g17260

[0030] 1. GWAS analysis of rice flooding tolerance traits and identification of the flooding tolerance gene LOC_Os06g17260

[0031] 1. Test materials

[0032] In this study, 300 japonica rice accessions were selected from natural populations as the GWAS study population. These accessions were widely sourced and representative. All seeds used in the study were dried in an oven at 45°C for 7 days after harvest to break dormancy.

[0033] 2. Genotype analysis

[0034] The genotype data were processed based on the 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 named "Plink" was used to check the missing rate and remove samples with high missing rates, retaining minor allele frequency (MAF)>0.05, minimum genotype quality of 0.4 and r 2 A total of 711,268 high-quality polymorphic SNPs were screened for SNPs with a threshold greater than 0.4. SNP data were sorted using RStudio Server and Linux system, and PCA principal component analysis was performed. Gene annotation was completed by calling the "java" program in the Linux system.

[0035] 3. Phenotypic identification of flooding 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, cover 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, and then immerse the seeds in a glass filled with sterile water with a depth of 15 cm. Place it in a germination room for cultivation (light / darkness, 16h / 8h, relative humidity 70%, constant temperature 26℃). After 7 days of cultivation, use a ruler to measure the shoot length and root length. In this study, 10 plants of each germplasm were used for evaluation of 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. The general linear model was used, and the critical p value of GLM was set (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 of the MSU Rice Genome Annotation Project Version 7 (http: / / rice.plantbiology.msu.edu / ), genes related to flooding tolerance were searched 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 in the interval 9801755-10001755: LOC_Os06g17090, LOC_Os06g17120, LOC_Os06g17140, LOC_Os06g17220 and LOC_Os06g17260. It is known that OsUGT75A regulates the length of coleoptiles and the flooding tolerance of rice germination by glycosylation-mediated reduction of free ABA and JA levels; at the same time, OsUGT75A may also promote the elongation of leaf sheaths by regulating the content of free ABA and JA, thereby promoting the germination of deeply sown rice seeds.

[0040] 2. Haplotype analysis of candidate genes

[0041] According to the variation of SNPs in the coding region of each gene, the haplotypes of the above five annotated genes were analyzed with the help of RStudio Server, and the significance of the differences in shoot length and root length between the main haplotypes consisting of more than 15 lines was tested. The results showed that LOC_Os06g17260 had two main 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 Hap1 and Hap2. Hap1 showed a longer shoot and was the dominant haplotype ( Figure 4 ).

[0042] Furthermore, the present invention has developed a molecular marker based on the above SNP variation, which amplifies a DNA fragment containing a specific SNP site through a pair of specific primers and distinguishes different haplotypes through sequencing, thereby realizing 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: Gene knockout verification of LOC_Os06g17260

[0046] 1. Construction of LOC_Os06g17260 knockout line

[0047] CRISPR / Cas9 was used to construct a knockout line (KO260) based on the wild-type ZH11 background for gene function verification. The target sequence was 5'-GCCGGCGCGCCAGGCAGCCCCGG-3', and the primer sequences F: 5'-ACGATACCTCTG GCCCGAC-3', R: 5'-GCCATACAGCCCAGACGC-3'.

[0048] II. Phenotypic identification of submergence tolerance of KO260 during germination

[0049] 1. Screening of KO260 homozygous strains

[0050] Young leaves of each KO260 plant were taken and genomic DNA was extracted according to the cetyltriethylammonium bromide (CTAB) method of Doyle and Dickson (1987). Primers were designed according to the target site, with the primer sequences A: 5'-ACGATACCTCTGGCCCGAC-3', B: 5'-GCCATACAGCCCAGACGC-3', and amplified by PCR.

[0051] The PCR amplification reaction system was 20 μl, including 1 μl DNA template, 1 μl (10 μM, F+R) primer, 10 μl 2×Buffer, 5 μl dNTPs (2 mM), 0.5 μl KOD FX and 2.5 μl ddH2O; the amplification reaction was carried out on a Professional Thermocycler (Biometra Inc.) amplification instrument, and the reaction program was: 94°C for 7 min; 98°C for 10 sec, 59°C for 30 sec, 68°C for 40 sec (1 min for a product length of 1 Kb), 68°C for 7 min, 34 cycles. The PCR product was detected by 1.5% agarose gel electrophoresis (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 flooding tolerance during the germination period according to the method in step 1 of Example 1, and a control treatment group was 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 gene for flooding tolerance during rice germination LOC_Os06g17260 , characterized in that, Its DNA sequence is shown in SEQ ID No. 1, or is its functional equivalent or homologous gene.

2. The gene according to claim 1 LOC_Os06g17260 , characterized in that, 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 major haplotypes, Hap1: TAC, Hap2: GCT.

3. A method for identifying or assisting in identifying a molecular marker for flooding tolerance in rice during the germination period, characterized in that: The molecular markers are based on LOC_Os06g17260 Specific SNP variation of the gene is designed to detect different haplotypes of the gene, among which the lines carrying Hap1 show stronger flooding tolerance, so as to identify or assist in the identification of flooding tolerance of rice.

4. The molecular marker according to claim 3, characterized in that It includes a pair of specific primers to amplify the DNA fragment containing the specific SNP site and distinguish different haplotypes through sequencing.

5. The molecular marker according to claim 4, characterized in that 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'.

6. A kit for identifying or assisting in identifying flooding tolerance of rice during germination, characterized in that: It includes a primer pair for detecting a molecular marker based on a LOC_Os06g17260 The specific SNP variation of a gene is designed to detect different haplotypes of that gene.

7. The kit according to claim 6, characterized in that 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'.

8. The kit according to claim 7, characterized in that Primer F and primer R were mixed in a 1:1 ratio.

9. A method for identifying or assisting in identifying flooding tolerance of rice during germination, characterized in that: The genomic DNA of the rice to be tested is used as a template, and the primer pair of the molecular marker described in claim 5 or 6 is used for PCR amplification, and then the flooding tolerance haplotype of the rice to be tested is identified by sequencing and sequence comparison to determine the flooding tolerance of the rice during germination.

10. 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 9 is used to obtain the flooding tolerance of the rice variety to be tested during the germination period, and the rice material to be tested having a specific flooding tolerance haplotype is selected for subsequent breeding.

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