Application of Oryza longistaminata OlGS1 and OlEXPB1 genes in self-incompatible restorer line

Abstract

The invention discloses application of OlGS1 and OlEXPB1 genes of oryza longistaminata in a self-incompatible restorer line, and relates to the technical field of rice cultivation and seed production, the key points of the technical scheme are as follows: KI-I2, pollen tube in-vivo germination and other tests prove that the physical properties of male and female gametes of oryza longistaminata are normal, the growth of pollen tubes is hindered during selfing, and the incompatible restorer line is incompatible with oryza longistaminata. The pollen tube grows normally during outcrossing. And screening out a candidate gene OlGS1 causing the self-incompatibility of the oryza longistaminata by combining cytology with RNA-seq. The interaction gene OlEXPB1 is screened from a wild oryza longistaminata anther cDNA library by taking OlGS1 as a bait. Gene knockout verification shows that the growth of a mutant pollen tube on a stigma is hindered, so that infertile rice selfing is caused. A self-incompatible restoring line containing OlGS1 and OlEXPB1 is screened from an RIL population by molecular marker-assisted selection and is matched with hybrid rice backbone parents Y58S, Xinan S and the like which are popularized in a large area in production, and results show that introduction of a self-incompatible gene in two environments does not affect the heterosis of the combination of the self-incompatible gene.

Description

Technical Field

[0001] The invention relates to the technical field of rice breeding, and more particularly to the application of Oryza longistaminata OlGS1 and OlEXPB1 genes in self-incompatibility restorer lines. Background Art

[0002] Self-incompatibility (SI) is a reproductive mechanism in higher plants that prevents inbreeding by inhibiting self-pollination. Self-incompatible plants can produce functional male and female gametes, but cannot produce seeds after self-pollination (Pandey, 1960). Based on genetic behavior, self-incompatibility can be divided into two types: sporophytic self-incompatibility (SSI) and gametophytic self-incompatibility (GSI) (De Nettancourt, 1997; McCubbin and Kao, 2000; Qu et al., 2015). SSI is mainly found in plants such as Cruciferae and Asteraceae (Dickinson, 1995; Iwano et al., 2015), while GSI is mainly found in plants such as Solanaceae, Rosaceae, Papaveraceae and Poaceae (Liu et al., 2014; Liang et al., 2020). The genetic mechanism of SSI depends on signal transduction to control single or multiple genetic loci between pollen and stigma, resulting in mature pollen grains carrying self-incompatibility signal molecules secreted by the tapetum, which manifests as pollen grains not germinating or pollen tubes coiling on the stigma (Hiscock and Tabah, 2003).

[0003] Previous studies have reported that genes such as SRK, SLG, SP11, SCR, ARC, MLPK, EXO70A1, THL1, and MOC are involved in the plant SSI pathway (Chen et al., 2017; Cabrillac et al., 2001; Takayama et al., 2001; Kakita et al., 2007). The genetic mechanism of GSI is mainly controlled by a single site in the S-locus of the vegetative pollen tube cells. Previous studies have reported that the S-RNase gene in this locus causes GSI, and nearly 100 S-RNase genes have been isolated from various plants such as Solanaceae and Rosaceae (Hu et al., 2014). However, Poaceae plants are typical double-factor self-incompatibility, which involves two unlinked S and Z genetic loci (Eaves et al., 2014). LpSDUF247 is a confirmed self-incompatibility SZ gene in rye (Shinozuka et al., 2010). Comparative genomics revealed that OlSS1 and OlSS2 in longistaminata wild rice are homologous to LpSDUF247 (Lian et al., 2021). Cytological studies on pollen tube inhibition in the GSI system have been carried out. It is generally believed that most incompatible pollen in this system are not affected in germination, can produce pollen tubes and pass through the stigma and grow to the style, but stop growing at different parts (Niu Junhai et al., 2006). With the deepening of research, it has been found that there are some factors in the gametophyte self-incompatibility reaction process that are not encoded by S-RNase genes but are necessary for the self-incompatibility reaction (Liu Suling et al., 2014).

[0004] In recent years, studies have shown that mutations in N-acetylglutamate kinase (NAGK) in Arabidopsis lead to early embryo sac development and pollen tube elongation. Studies have also found that arginine is completely synthesized by embryo sac tissue cells themselves, which shows that the arginine synthesis pathway plays an important role in the interaction between male and female gametes, pollen tube elongation, and embryo sac development (Jie et al., 2017). Expansin can promote cell wall expansion and is a protein with a wide range of effects on plant growth. It is currently believed that Expansin is involved in almost the entire development process of plants. In addition to its function of enlarging cells, Expansin plays an important role in promoting plant vegetative growth, root growth, pollen tube growth, and fruit ripening. Herbaceous Class I pollen allergens are also a type of Expansin protein specifically expressed by plant pollen. This protein relaxes the cell walls of the stigma and style, making it easier for pollen tubes to pass through the style and enter the ovary (Shcherban et al., 1995).

[0005] Hybrid rice has high individual heterozygosity, and the hybrid offspring have separated traits, so seeds need to be produced every year. "The country is based on agriculture, and agriculture is based on seeds first." The improvement and optimization of hybrid rice seed production methods have played a leading role in promoting the development of my country's hybrid rice industry. Whether it is three-line hybrid rice or two-line hybrid rice, there is a situation of high labor cost and low production efficiency in the process of hybrid rice seed production, which seriously restricts the development of my country's hybrid rice industry. Hybrid rice mixed sowing and mixed harvesting seed production technology can realize the simplification of seed production. In recent years, many research teams have conducted a series of studies on mixed sowing and mixed harvesting seed production technology, mainly through color sorting (He Libin et al., 2001), grain size difference screening method (Zhou Jieqiang et al., 2021) and transgenic label removal (Yuan Longping, 2016) and other methods to separate hybrids from paternal seeds in hybrid rice seed production, but for various reasons, these methods have not yet been applied on a large scale in hybrid rice seed production practice. Summary of the invention

[0006] The purpose of the present invention is to provide the application of OlGS1 and OlEXPB1 genes of Oryza longistaminata in self-incompatibility restorer lines, to clarify the molecular regulation mechanism of self-incompatibility of Oryza longistaminata, and further to break through the technical bottleneck of mixed sowing and mixed harvesting in the hybrid rice seed production process through biotechnology means, to improve the mixed sowing and mixed harvesting seed production technology system, and to reduce the production cost of hybrid rice seeds.

[0007] The above technical purpose of the present invention is achieved through the following technical scheme: application of OlGS1 and OlEXPB1 genes of longistaminata wild rice in self-incompatibility restorer lines, on the basis of obtaining gene functions of OsGS1 and OsEXPB1 knockout mutants osgs1-KO and osexpb1-KO, screening self-incompatibility restorer lines containing OlGS1 and OlEXPB1 from longistaminata wild rice RIL population, which can not only enable hybrid rice restorer lines to obtain self-incompatibility characteristics, but also do not affect the hybrid vigor and other agronomic traits of their mating offspring. Therefore, the longistaminata wild rice RIL introduction line containing OlGS1 and OlEXPB1 genes is more suitable for the breeding of self-incompatibility restorer lines and mixed sowing and mixed harvesting production practices.

[0008] In summary, the present invention has the following beneficial effects:

[0009] 1. Oryza longistaminata is an excellent germplasm resource material originating from Africa, with stable self-incompatibility. As an intraspecific reproductive barrier, self-incompatibility is of great significance in promoting the formation of interspecific reproductive barriers and species differentiation. Using Oryza longistaminata as a genetic research material, new self-incompatibility OlGS1 and OlEXPB1 gene loci were identified, which is the first new gene cloned in Oryza longistaminata for the interaction between male and female gametophytes. At the same time, revealing the molecular mechanism of its self-incompatibility can further enrich the theoretical basis of plant self-incompatibility.

[0010] 2. Using the mixed sowing and mixed harvesting technology of hybrid rice sterile lines and restorer lines to reduce the labor cost of hybrid rice seed production is a feasible way, but how to effectively solve the self-sterility of restorer lines used for seed production has always been a problem in the rice breeding industry. Using the stable self-incompatibility gene of wild rice longistaminata to cultivate self-incompatibility restorer lines may be an important method to solve the hybrid rice mixed sowing and mixed harvesting seed production technology. Based on the analysis of the function and molecular regulation mechanism of the self-incompatibility gene of wild rice longistaminata, this project evaluates its utilization value in improving the fertility of restorer lines, which will provide new gene resources for hybrid rice breeding upgrades.

[0011] Elucidating the molecular regulatory mechanism of self-incompatibility of Oryza longistaminata and then using biotechnology to break through the technical bottleneck of mixed sowing and mixed harvesting in hybrid seed production and reduce seed production costs will be of great significance to promoting the sustainable and healthy development of my country's hybrid rice seed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the male gamete fertility characteristic of the longistaminata wild rice in the embodiment of the present invention;

[0013] Note: A. Plant phenotype of Oryza longistaminata; B. Panicle phenotype of Oryza longistaminata; C. Pollen I of Oryza longistaminata 2 -KI staining; D. TTC staining of Oryza longistaminata pollen; E. Elongation of pollen tube on stigma after outcrossing of Oryza longistaminata 9311 and ZH11; F. Mature embryo sac after outcrossing of Oryza longistaminata 9311 and ZH11;

[0014] Figure 2 is the female gamete fertility characteristic of the longistaminata wild rice in the embodiment of the present invention;

[0015] Note: a. Panicle phenotype of Oryza longistaminata at flowering stage; b. Flower organ phenotype of Oryza longistaminata; c. Aniline blue staining shows the germination of pollen tubes on the stigma after self-pollination of Oryza longistaminata. The germination times are 5 min, 0.5 h, 1 h, 2 h and 4 h after pollination, respectively. Bar = 50 um; d. The ovary is transparent as a whole, showing the development process of the embryo sac of Oryza longistaminata. The development stages are megaspore mother cell, dyad, tetrad, mononuclear embryo sac, dinuclear embryo sac, tetranuclear embryo sac, octanuclear embryo sac and mature embryo sac, respectively. Bar = 50 um;

[0016] Figure 3 is a transcriptome analysis of the self-incompatibility trait of Oryza longistaminata in an embodiment of the present invention;

[0017] Note: a, Cluster heat map of Pearson correlation coefficients before and after self-pollination between Oryza longistaminata and cultivated rice; b, PCA principal component analysis before and after self-pollination between Oryza longistaminata and cultivated rice; c, Cluster heat map of module genes before and after self-pollination between Oryza longistaminata and cultivated rice; d, 27 differentially expressed genes significantly downregulated before and after self-pollination between Oryza longistaminata and cultivated rice; e, qRT-PCR verification of 8 candidate genes in the transcriptome;

[0018] Figure 4 The expression pattern and subcellular localization characteristics of Oryza longistaminata in the embodiment of the present invention;

[0019] Note: AP OlGS1 ::GUS staining verification of GUS transgenic plants; B. Subcellular localization of OlGS1 protein; CP OlGS1 :: Subcellular stable transformation verification of OlGS1-GFP transgenic plants;

[0020] Figure 5 The method is to screen the yeast library of the male ligand protein of Oryza longistaminata in the embodiment of the present invention;

[0021] Note: a. Toxicity and self-activation analysis of recombinant plasmids in yeast two-hybrid assay; b. Verification results of four-deficient plates for positive clones in yeast library screening; c. BLAST comparison analysis results of male ligand protein of Oryza longistaminata;

[0022] Figure 6 It is the interaction verification of the key protein of Oryza longistaminata in the embodiment of the present invention;

[0023] Note: a. Yeast two-hybrid dot-to-dot verification of OlGS1 and OlEXPB1; b. Bimolecular fluorescence complementation (BiFC) verification of OlGS1 and OlEXPB1; c. Co-IP in vivo interaction verification of OlGS1 and OlEXPB1; d. Pull-down in vitro interaction verification of OlGS1 and OlEXPB1;

[0024] Figure 7 is the haplotype analysis of the candidate gene OsGS1 in the embodiment of the present invention;

[0025] Note: A. Gene structure and haplotype variation sites of OsGS1; B. Number of materials with different haplotypes of OsGS1 in five subpopulations, including Xian, Geng, Basmati, Aus and admix; C. Number of materials with different haplotypes of OsGS1 in five subpopulations, including Xian, Geng, Or1, Or2 and Or3; D. Number of materials with different haplotypes of OsGS1 in six subpopulations, including local varieties and modern varieties with different backgrounds; E, F, G and H. Nucleotide diversity (π), inter-population fixation index (Fst), Tajima's D value and cross-population composite likelihood ratio test (XP-CLR) score of OsGS1 and OsEXPB1;

[0026] Figure 8 The OsGS1 target gene knockout site design and mutation sequence analysis in the embodiment of the present invention;

[0027] Note: Schematic diagram showing the structure of the OsGS1 gene with the Crispr-cas9 target site. Exons are represented by black boxes. The figure below shows the alignment of the WT and osgs1-9(ZH11) sequences containing the Crispr-cas9 target site. The osgs1-9(ZH11) mutant contains a 1 bp deletion (-, red font);

[0028] Fig. 9 The OsEXPB1 target gene knockout site design and mutant sequence analysis in the embodiments of the present invention;

[0029] Note: Schematic diagram showing the structure of the OsEXPB1 gene with the Crispr-cas9 target site. Exons are represented by black boxes. The figure below shows the alignment of the WT and osexpb1-7(ZH11) sequences containing the Crispr-cas9 target site. The osexpb1-7(ZH11) mutant contains a 1 bp base insertion (A, red font);

[0030] Fig.10 The phenotype verification and pollen tube growth characteristics of the transgenic knockout materials osgs1-KO and osexpb1-KO in the embodiments of the present invention;

[0031] Note: a. Plant phenotype of transgenic knockout material osgs1-KO; b. Ear and grain type phenotype of transgenic knockout material osgs1-KO; c. Pollen tube growth on the stigma of transgenic knockout material osgs1-KO at heading stage, Bar=5cm; d. Plant phenotype of transgenic knockout material osexpb1-KO; e. Ear and grain type phenotype of transgenic knockout material osexpb1-KO; f. Pollen tube growth on the stigma of transgenic knockout material osexpb1-KO at heading stage, Bar=5cm; g. Seed setting rate of transgenic knockout material osgs1-KO; h. Relative expression level of OsGS1 gene in transgenic knockout material osgs1-KO; i. Seed setting rate of transgenic knockout material osexpb1-KO; j. Relative expression level of OsEXPB1 gene in transgenic knockout material osexpb1-KO;

[0032] Fig.11 It is the male and female gamete fertility characteristics of the homozygous strains of OlGS1 and OlEXPB1 gene knockout in the embodiment of the present invention;

[0033] Note: a. I of wild-type ZH11 2 -KI staining, TTC staining, pollen germination rate and scanning electron microscopy of pollen grains; b. I of OlGS1 gene knockout homozygous strain 2 -KI staining, TTC staining, pollen germination in vitro and scanning electron microscopy of pollen grains; c. I of the homozygous OlEXPB1 gene knockout line 2 -KI staining, TTC staining, pollen germination in vitro and scanning electron microscopy of pollen grains; d. Pollen germination rate of homozygous strains of OlGS1 gene knockout; e. Pollen germination rate of homozygous strains of OlEXPB1 gene knockout; f. Dynamic development process of mature embryo sac of wild-type ZH11, OlGS1 and OlEXPB1 gene knockout homozygous strains;

[0034] Fig.12 It is a comparison of agronomic traits between the transgenic knockout plants and the wild type in the examples of the present invention.

[0035] Note: A. Plant height of WT (ZH11) and osgs1-KO, osexpb1-KO, osgs1 / osexpb1-KO knockout lines at the mature stage; B. Ear length of WT (ZH11) and osgs1-KO, osexpb1-KO, osgs1 / osexpb1-KO knockout lines at the mature stage; C. Tiller number of WT (ZH11) and osgs1-KO, osexpb1-KO, osgs1 / osexpb1-KO knockout lines at the mature stage; D. E. Primary branches of osgs1 / osexpb1-KO knockout lines at the mature stage; E. Secondary branches of WT (ZH11) and osgs1-KO, osexpb1-KO, osgs1 / osexpb1-KO knockout lines at the mature stage; F. Total number of grains per ear of WT (ZH11) and osgs1-KO, osexpb1-KO, osgs1 / osexpb1-KO knockout lines at the mature stage; There was no significant difference between WT and osgs1-KO, osexpb1-KO, osgs1 / osexpb1-KO, ns indicates P>0.05.

[0036] Fig.13 It is a preliminary breeding evaluation of the self-incompatible introgression line in the embodiment of the present invention;

[0037] Note: a. Plant types of the four self-incompatible introgression lines; b. Ear types of the four self-incompatible introgression lines; cf. Plant types of the offspring of the self-incompatible introgression lines mated with Y58S, Xin'an S, Jing 4155S, and Longke 638S; cf. Comparison of ear types and single-plant yield of the offspring of the self-incompatible introgression lines mated with Y58S, Xin'an S, Jing 4155S, and Longke 638S; k. Mixed sowing and mixed harvesting seed production model diagram.

[0038] Fig.14 This is the heterosis analysis of the offspring of the self-incompatible introgression lines in the embodiments of the present invention.

[0039] Note: a. Comparison of heterosis of yield traits (effective tillering, total number of grains per ear, 1000-grain weight, seed setting rate, and yield per plant) of offspring of self-incompatible introgression lines mGS1 / 9311 and 9311 parents respectively crossed with Y58S, Xin'an S, Jing 4155S, and Longke 638S under Jiangxi environment; b. Comparison of heterosis of yield traits (effective tillering, total number of grains per ear, 1000-grain weight, seed setting rate, and yield per plant) of offspring of self-incompatible introgression lines mGS1 / 9311 and 9311 parents respectively crossed with Y58S, Xin'an S, Jing 4155S, and Longke 638S under Guangdong environment Comparison; c. Comparison of heterosis of other agronomic traits (plant height, ear length, primary stalk, secondary stalk) in the offspring of the self-incompatible introgression lines mGS1 / 9311 and 9311 parents mated with Y58S, Xin'an S, Jing 4155S, and Longke 638S respectively under Jiangxi environment; d. Comparison of heterosis of other agronomic traits (plant height, ear length, primary stalk, secondary stalk) in the offspring of the self-incompatible introgression lines mGS1 / 9311 and 9311 parents mated with Y58S, Xin'an S, Jing 4155S, and Longke 638S respectively under Guangdong environment; ns indicates P>0.05. DETAILED DESCRIPTION

[0040] The following is combined with Figure 1-14 The present invention is further described in detail. In the following text, "OlGS1" and "OlEXPB1" written in italics are gene names, and those written in non-italics are the names of proteins produced by the expression of the gene.

[0041] Example 1: Cytological analysis and gene mapping of the self-incompatibility trait of Oryza longistaminata

[0042] 1. Verification of the fertility of male gametophytes of Oryza longistaminata

[0043] I 2 -KI staining detected that the pollen color of longistaminata wild rice was dark brown, and the pollen fertility was 97.8%±1.23 (n=800). The results showed that the fertility of male gametes was completely normal. Figure 1 As shown in C. TTC staining detected that the pollen color was red or pink, and the pollen activity was 96.2% ± 2.54 (n = 630). The results showed that the pollen activity was completely normal. Figure 1 As shown in D. The pollen tube elongation test of the cross between 9311 and ZH11 of Oryza longistaminata showed that pollen could germinate on the stigma 6 hours after pollination and extend into the ovary through the style, indicating that the pollen tube elongated normally on the stigma. Figure 1 As shown in E; after the mature embryo sac is fertilized, the pollen tube has reached the micropyle of the ovule of 9311 and ZH11. Figure 1 As shown in center F, the results showed that pollen viability and post-fertilization elongation of Oryza longistaminata were normal.

[0044] 2. Fertility verification of female gametophytes and pollen tube growth test of Oryza longistamica

[0045] Phenotypic observations during the flowering period showed that the anthers of longistambar wild rice opened normally and the pollen was scattered. Figure 2 As shown in a. Observation under a stereomicroscope shows that the male and female flower organs of long-staminate wild rice develop synchronously, as shown in Figure 2 As shown in b. After 5 minutes of self-pollination with bagging, the pollen pores have protruded; after 30 minutes of self-pollination, the pollen tubes have grown, but the length of the pollen tubes has not reached the length of the pollen diameter; after 1 hour of self-pollination, the pollen tubes continue to elongate, and the length of the pollen tubes has exceeded the diameter of the pollen; after 4 hours of self-pollination, the elongation of the pollen tubes on the style is inhibited and stops at the branch of the feathery stigma. The results show that the pollen of wild rice can germinate but the growth of the pollen tubes is subsequently blocked, which belongs to the self-incompatibility of the gametophyte, such as Figure 2 As shown in c. The female gametophyte of Oryza longistaminata develops normally and can produce eight-nucleated cells, such as Figure 2 As shown in middle d.

[0046] 3. RNA-seq dynamic developmental screening of the female receptor gene OlGS1 in the self-incompatibility of Oryza longistaminata

[0047] In order to identify the candidate genes that determine the self-incompatibility of longistaminata wild rice, this study selected gene clusters in which the dynamic gene expression of longistaminata wild rice showed a downward trend in 2mm, 6mm, 7mm and 8.5mm spikelet samples, while the dynamic gene expression of cultivated rice showed an upward trend. It was found that there were genes with significant differences between the 8.5mm spikelet samples of longistaminata wild rice and cultivated rice. The cluster analysis results showed the top 27 differentially expressed genes between longistaminata wild rice and cultivated rice, such as Figure 3 As shown in ad. Among them, 8 genes were annotated by NCBI as being highly expressed in floral organs. These genes are involved in metabolic pathways such as arginine synthesis, brassinolide synthesis, and glyoxylate cycle. We selected unknown proteins in these 8 fertility-related metabolic pathways as candidate genes. In order to verify the accuracy of the transcriptome data, we selected 9 candidate genes for qRT-PCR verification. The results showed that the qRT-PCR results of most candidate genes were basically consistent with the transcriptome data. Among them, it has been reported that arginine is related to the interaction between male and female gametophytes, pollen tube elongation, and embryo sac development. Therefore, we screened out 2 candidate genes, named OsGS1, as Figure 3 As shown in figure e.

[0048] 4. Expression pattern and subcellular localization characteristics of the female receptor gene OlGS1 in Oryza longistaminata

[0049] The GUS staining results showed that the signal appeared in the feathery stigma and style, indicating that the site of action of the OlGS1 gene function may be located in the feathery stigma and style of the pistil, e.g. Figure 4As shown in A. OlGS1-PAN580 recombinant vector was used for transient expression in rice protoplasts. The results showed that the fusion protein OlGS1::GFP detected a green fluorescence signal, and the co-transfected fusion protein mKATE::RFP detected a red fluorescence signal. The overlapped field of OlGS1 and mKATE, which is known to be localized on the rough endoplasmic reticulum, appeared yellow, indicating that OlGS1 is localized on the rough endoplasmic reticulum, indicating that OlGS1 is a secretory protein. Figure 4 As shown in B. The rice callus was transformed with a eukaryotic expression vector to obtain a stable transgenic plant. The results showed that the OlGS1 gene was specifically expressed in the rice stigma. Figure 4 As shown in C.

[0050] 5. Screening of yeast library of male ligand genes of wild rice and verification of interaction

[0051] The negative control, positive control and self-activation test strains can all grow on DDO and TDO plates, indicating that the plasmid was successfully co-transformed into yeast cells. However, only the positive control can grow on QDO / X plates, and the negative control and self-activation test groups cannot grow on QDO / X plates, indicating that OlGS1 does not self-activate and is not toxic to yeast, and subsequent library screening experiments can also be performed. Using the recombinant clone of the OlGS1 gene cDNA as bait, the interacting proteins were screened from the yeast library of the anther tissue of Oryza longistaminata, and the positive plasmid DNA sequencing in the yeast colony was subjected to BLAST comparison analysis. After removing the duplicates, there were 10 different protein encoding genes. The function prediction showed that Expansin-B4 may be related to the relaxation of the pollen tube and style wall cells, and was named OlEXPB1. Figure 5 shown.

[0052] The full-length cDNA of OlEXPB1 was amplified and yeast two-hybridization was performed to verify the interaction between OlGS1 and OlEXPB1. Figure 6 As shown in a. Through bimolecular fluorescence complementation verification, the visualization results show that OlGS1 and OlEXPB1 interact with each other in the cell membrane and participate in the interaction between male and female gametes, as shown in Figure 6 As shown in b. By constructing eukaryotic expression transgenic lines, Co-IP results showed that there was an interaction between OlGS1 and OlEXPB1 in rice, such as Figure 6 As shown in c. By constructing a prokaryotic expression vector and performing protein homozygosity, the pull-down results showed that there was a direct interaction between OlGS1 and OlEXPB1, as shown in Figure 6 As shown in middle d.

[0053] 6. Haplotype analysis of candidate gene OlGS1

[0054] Haplotypes in 3K rice germplasm resources were classified based on all SNPs (ignoring synonymous SNPs) in the CDS region of genes downloaded from the RFGB v2.0 database (Wang et al., 2020). Haplotypes that appeared in at least 30 rice varieties were defined as major haplotypes. Population genetic analysis was performed on 3359 japonica rice, 5295 indica rice, and 412 wild rice genome sequences from the Rice Super-Population Variation Map database (Wang et al., 2023). For the 200 kb regions upstream and downstream of the gene, VCFtools software (v0.1.16) was used to calculate the nucleotide diversity (π) and inter-population fixation index (Fst) of each window with a window of 5 kb and a step size of 1 kb, and Tajima's D of each window was calculated with a window of 5 kb; the python package XPCLR was used to calculate the cross-population composite likelihood ratio test (XP-CLR) score of each window with a window of 5 kb, a step size of 1 kb and an ld of 0.95.

[0055] Haplotype analysis of the candidate gene OsGS1 showed that only 6 haplotypes had more than 10 copies of each haplotype, including the representative self-compatible haplotypes Hap1, Hap2, Hap3, Hap5, Hap6 and the self-incompatible haplotype Hap4. In local varieties, 86.7% of Hap1 came from indica rice, and 55.7% of Hap2 came from japonica rice; in contemporary varieties, 83.9% of Hap1 came from indica rice, and 65.2% of Hap2 came from japonica rice; in addition, 3.1% of Hap4 came from wild rice. The results showed that OsGS1 partially explained the variation in self-incompatibility between indica and japonica rice subpopulations and wild rice.

[0056] Example 2 Creation of OsGS1 and OsEXPB1 transgenic knockout materials

[0057] Gene editing of designed targets of OsGS1 and OsEXPB1 in ZH11 background ( Figure 4 A and B), sequencing results showed that the gene sequences of OsGS1 and OsEXPB1 in the above transgenic knockout lines had frameshift mutations, resulting in premature termination of protein translation, and cytological and fruiting rate phenotype identification could be performed. In this study, the crispr-cas9 technology was used to knock out the gene of Zhonghua 11 (ZH11) and T 2The first generation of transgenic plants were sequenced to screen homozygous plants, among which the homozygous mutant osgs1-9 (ZH11) had reduced fertility. The results showed that the first exon of OsGS1 had a G base deletion at +13nt upstream of the PAM sequence, resulting in premature termination of OsGS1 translation. Figure 8 shown.

[0058] CRISPR-Cas9 technology was further used to knock out the gene of ZH11 and T 2 The first generation of transgenic plants were sequenced to screen homozygous plants, among which the homozygous mutant osexpb1-7 (ZH11) had reduced fertility. The results showed that the first exon of OsEXPB1 had an A base inserted at +13nt upstream of the PAM sequence, resulting in premature termination of OsEXPB1 translation. Fig. 9 shown.

[0059] Example 3 Functional verification of OsGS1 and OsEXPB1 gene knockout materials

[0060] After knocking out the OsGS1 and OsEXPB1 genes, the results showed that the plants of osgs1-KO and osexpb1-KO knockout materials were completely sterile, such as Fig.10 As shown in a and c. Compared with the wild type, the panicle, grain, stigma and anther morphology of osgs1-KO and osexpb1-KO knockout materials were normal, as shown in Fig.10 As shown in Figures b and e. Aniline blue staining was performed on the germination of pollen tubes on the stigma after self-pollination. The results showed that the pollen tubes of wild-type ZH11 basically passed through the style 4 hours after flowering, while the pollen tube elongation of osgs1-KO and osexpb1-KO knockout materials stagnated, as shown in Figures c and f. The fruit set rate of osgs1-KO and osexpb1-KO was significantly lower than that of WT (ZH11), as shown in Figures c and f. Fig.10 As shown in g and i, the relative gene expression levels of osgs1-KO and osexpb1-KO were significantly lower than those of WT (ZH11). Fig.10 As shown in h and j. The results showed that gene knockout of OsGS1 and OsEXPB1 affected the germination of pollen tubes on the stigma, further leading to self-incompatibility.

[0061] In this study, the OlGS1 and OlEXPB1 gene knockout homozygous lines were isolated and purified. 2 -KI staining showed that the pollen color of osgs1-9 and osexpb1-7 was dark brown, and the male gamete fertility was normal. Fig.11 As shown in Figures ac; TTC staining of the homozygous knockout lines of OlGS1 and OlEXPB1 showed that the pollen color of osgs1-9 and osexpb1-7 was pink and the pollen vitality was normal, as shown in Figure 4. Fig.11 As shown in ac; the pollen germination of the homozygous strains of OlGS1 and OlEXPB1 gene knockout was carried out in vitro. The results showed that most of the pollen germinated and grew pollen tubes in osgs1-9 and osexpb1-7, and the length of the pollen tubes was far greater than the diameter of the pollen. The pollen tube germination was normal, as shown in Fig.11 As shown in Figure ac; Scanning electron microscopy was used to observe the pollen grains of the homozygous strains of OlGS1 and OlEXPB1 gene knockout. The results showed that the pollen grains of osgs1-9 and osexpb1-7 were normal in morphology, as shown in Figure 4. Fig.11 Compared with the wild type, there was no significant difference in pollen germination rate between osgs1-9 and osexpb1-7. Fig.11 As shown in d and e. The embryo sac of the homozygous knockout strains of OlGS1 and OlEXPB1 was observed to be transparent throughout the ovary. The results showed that the mature embryo sac could produce eight-nucleated cells and the female gametophyte developed normally, as shown in Fig.11 As shown in figure f.

[0062] Example 4 Effects of OsGS1 and OsEXPB1 on agronomic traits

[0063] To investigate whether knockout of OsGS1 and OsEXPB1 would have adverse effects on the normal growth and development of rice, we investigated the six main agronomic traits of plant height, panicle length, tiller number, primary stalk, secondary stalk, and total number of grains per panicle of the OsGS1 and OsEXPB1 knockout mutants osgs1, osexpb1, osgs1 / osexpb1 and their wild type ZH11 in the field at the Sanya transgenic base. The results showed that there were no significant differences in agronomic traits such as plant height, panicle length, tiller number, primary stalk, secondary stalk, and total number of grains per panicle between the transgenic knockout plants osgs1, osexpb1, osgs1 / osexpb1 and the wild type ZH11. The above results show that the knockout mutants of OsGS1 and OsEXPB1, osgs1 and osexpb1, can not only make the restorer line of hybrid rice acquire the characteristics of self-incompatibility, but also do not affect other agronomic traits of the male parent. Therefore, the knockout mutants of OsGS1 and OsEXPB1 are more suitable for the breeding of self-incompatibility restorer lines and mixed sowing and mixed harvesting production practices. Fig.12 shown.

[0064] Example 501 Evaluation of the breeding value of GS1

[0065] To test whether the functional loss of OsGS1 and OsEXPB1 can produce the sterility trait caused by self-incompatibility in cultivated rice, which is a prerequisite for the application of this gene in hybrid seed production, OlGS1 was introduced into 9311 ( Fig.13a, b). The self-incompatible introgression line mGS / 9311 was screened from the offspring of the high-generation recombinant inbred lines of these two genetic introgressions. Individuals carrying the OlGS1 mutation were sterile, and both male and female gametophytes developed normally, but the growth of pollen tubes on the stigma was blocked. The self-incompatible introgression line mGS / 9311 was further matched with the backbone female parents of the hybrid combination Y58S, Xin'an S, Jing 4155S, and Longke 638S that are currently widely promoted in production. The overall plant type of the hybrid combination had no significant difference from the combination promoted on a large area ( Fig.13 cj). In traditional hybrid seed production programs, traditional restorer lines need to be planted separately from male sterile lines, mainly for the purpose of better removing them from the field before large-scale mechanized harvesting of hybrids. In contrast, when using self-incompatible introductory lines as restorer lines, there is no need to remove restorer plant bodies due to hybrids produced by self-pollination of the restorer lines. At the same time, seeds of self-incompatible introductory lines can be mixed in a certain proportion and mechanized in field trials, or hybrids can be harvested directly from the field. Compared with traditional hybrid seed production, the mixed sowing and mixed harvesting seed production model significantly reduces labor costs mainly in transplanting, powder removal and weed removal, which are 20%, 4% and 8% respectively, and the total cost of the entire production process is reduced by about 30% ( Fig.13 k).

[0066] To further test the application of mGS / 9311 in hybrid rice mixed sowing and mixed harvesting production, field trials were conducted in the summer optimal growing season in Jiangxi and Guangdong, China. The hybrid vigor of yield traits and main agronomic traits of hybrid combinations, such as plant height, number of tillers, seed setting rate, total number of grains, 1000-grain weight, yield per plant, etc., was compared and analyzed. The results showed that after the introduction of the longistaminata wild rice self-incompatibility gene OlGS1, the combination of the self-incompatible introductory line mGS / 9311 and its parent 9311 had no significant differences in yield traits (effective tillers, total number of grains per panicle, 1000-grain weight, seed setting rate, yield per plant) and main agronomic traits (plant height, panicle length, primary stalks, secondary stalks) in Jiangxi and Guangdong environments. Fig.14 ad). The results show that the introduction of the self-incompatibility gene of longistaminata wild rice into the restorer line will not affect the heterosis of hybrid rice. In summary, these natural germplasm resources are suitable for hybrid rice mixed sowing and mixed harvesting seed production.

[0067] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. Application of OlGS1 and OlEXPB1 genes of Oryza longistaminata in self-incompatibility restorer lines.

2. The use of the Oryza longistaminata OlGS1 and OlEXPB1 genes in self-incompatibility restorer lines according to claim 1, characterized in that: The OlGS1 and OlEXPB1 genes enable the hybrid rice restorer line to obtain self-incompatibility characteristics through knockout mutants osgs1-KO and osexpb1-KO.

3. The use of the Oryza longistaminata OlGS1 and OlEXPB1 genes in self-incompatibility restorer lines according to claim 2, characterized in that: The OlGS1 and OlEXPB1 gene markers are used for auxiliary selection in the longistaminata wild rice RIL population, which is suitable for the cultivation of self-incompatible restorer lines and mixed sowing and mixed harvesting production practices.

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