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CN-121992007-A - Method for producing rice haploid and application of method in breeding

CN121992007ACN 121992007 ACN121992007 ACN 121992007ACN-121992007-A

Abstract

The invention discloses a method for producing rice haploid and application thereof in breeding, which is characterized in that a rice haploid induction line is obtained by editing one or more of OsFEB1, osFEB2 and OsFEB3 genes in rice to lose the functions of OsFEB, osFEB2 and/or OsFEB3 genes, and the generation of a sexual reproduction induced haploid is carried out, wherein the gene sequence of OsFEB1 is shown as SEQ ID NO.1, the gene sequence of OsFEB2 is shown as SEQ ID NO.2, and the gene sequence of OsFEB3 is shown as SEQ ID NO. 3. The haploid induction line obtained by the method provided by the invention has the capability of inducing generation of haploid plants for breeding. Combining RHG1 haploid induction system with MIME system, artificially creating apomictic material to fix rice heterosis.

Inventors

  • LV QIMING
  • ZHANG XIULI
  • ZHAO BINGRAN
  • SUN CHUANQING
  • WANG QING
  • LI YUANJIE
  • XIONG SHUO
  • ZHAO YAN
  • XIA YUMEI

Assignees

  • 湖南杂交水稻研究中心

Dates

Publication Date
20260508
Application Date
20241108

Claims (8)

  1. 1. A method for producing rice haploid, which is characterized in that a rice haploid induction line is obtained by modifying one or more of OsFEB1, osFEB2 and OsFEB3 genes in rice to lose the functions of the OsFEB, osFEB2 and/or OsFEB3 genes, the rice haploid induction line induces the generation of haploid through sexual reproduction, the gene sequence of OsFEB1 is shown as SEQ ID NO.1, the gene sequence of OsFEB2 is shown as SEQ ID NO.2, and the gene sequence of OsFEB is shown as SEQ ID NO. 3.
  2. 2. The method of claim 1, wherein the OsFEB, osFEB2 and/or OsFEB gene is disabled by knocking out one or more of OsFEB, osFEB2 and OsFEB3 genes in rice by CRISPR/Cas9 system.
  3. 3. The method according to claim 2, characterized in that it comprises the steps of: (1) Designing primers according to gene sequences of OsFEB1, osFEB2 and/or OsFEB, and constructing a CRISPR/Cas9 vector A with OsFEB, osFEB2 and/or OsFEB3 knocked out genes; (2) Transforming the CRISPR/Cas9 vector A into rice to obtain a double-allele homozygote knockout T0 generation plant of which the knockouts OsFEB, osFEB2 and/or OsFEB3 are knocked out; (3) And sowing the T0 generation plants, and obtaining the rice haploid induction line by selfing.
  4. 4. The method of claim 3, wherein the primers designed according to the OsFEB1 gene sequence comprise FEB1-1F and FEB1-1R, the primers designed according to the OsFEB2 gene sequence comprise FEB1-2F and FEB1-2R, and the primers designed according to the OsFEB3 gene sequence comprise FEB1-3F and FEB1-3R; the gene sequence of the FEB1-1F is shown as SEQ ID NO. 4; The gene sequence of the FEB1-1R is shown in SEQ ID NO. 5; The gene sequence of the FEB2-1F is shown as SEQ ID NO. 6; the gene sequence of the FEB2-1R is shown in SEQ ID NO. 7; The gene sequence of the FEB3-1F is shown in SEQ ID NO. 8; The gene sequence of the FEB3-1R is shown in SEQ ID NO. 9.
  5. 5. The method according to any one of claims 1 to 4, wherein said rice is indica rice.
  6. 6. Use of a rice haploid obtainable by a method according to any one of claims 1 to 5 in breeding.
  7. 7. The application according to claim 6, characterized in that the method of application comprises the steps of: S1, designing a primer according to gene sequences of PAIR1, osREC and OsOSD1, and constructing a CRISPR/Cas9 carrier B which knocks out three genes of PAIR1, osREC8 and OsOSD 1; s2, co-transforming the CRISPR/Cas9 carrier A and the CRISPR/Cas9 carrier B into rice seeds to obtain double-allele homozygous knockout T0 generation plants; S3, selfing the T0 generation plant to obtain a T1 generation seed, so as to fix rice heterosis.
  8. 8. The use according to claim 7, wherein the primers designed according to the gene sequence of PAIR1 comprise PAIR1-1F and PAIR1-1R, the primers designed according to the gene sequence of OsREC comprise REC8-1F and REC8-1R, the primers designed according to the gene sequence of OsOSD1 comprise OSD1-1F and OSD1-1R; The gene sequence of the OSD1-1F is shown as SEQ ID NO. 10; The gene sequence of the OSD1-1R is shown as SEQ ID NO. 11; The gene sequence of PAIR1-1F is shown in SEQ ID NO. 12; the gene sequence of PAIR1-1R is shown in SEQ ID NO. 13; the gene sequence of REC8-1F is shown as SEQ ID NO. 14; The gene sequence of REC8-1R is shown as SEQ ID NO. 15.

Description

Method for producing rice haploid and application of method in breeding Technical Field The invention belongs to the technical fields of biotechnology and plant breeding, and particularly relates to a method for generating rice haploids and application of the method in breeding. Background Compared with the conventional rice, the hybrid rice generally has 20% of yield advantage, greatly contributes to guaranteeing the grain safety in China and even the world, and is recommended by grain and agriculture organizations in united nations as a first-choice technology for solving the grain problem in the world. However, the filial generation of the hybrid rice can be separated in character and cannot be directly reserved, and the annual seed production is needed, so that the production cost is greatly increased, the popularization and the application of the hybrid rice in a larger area are affected, and the full utilization of the hybrid vigor of the rice is also hindered. Only if the hybrid rice breeding technology is continuously innovated, the seed production cost is reduced, the popularity of the hybrid rice is improved, and more farmers and merchants can benefit from the technology. As early as 1987, yuan Longping institutions proposed that hybrid vigour fixation could be achieved by a first line hybrid rice based on apomixis. Apomixis of a plant refers to the way in which male and female gametes develop into new individuals without undergoing meiosis and fusion. The first line method is mainly to fix the heterosis of the hybrid rice F 1 generation by using apomixis mode, and is also considered as the highest strategic goal of hybrid rice breeding. The offspring of the apomictic material created by manpower can not have character separation, just like the conventional rice, a planter can seed and plant the apomictic material by himself, and the production cost of hybrid rice is greatly reduced. Thus, fixed rice vigor is developing as a leading edge of research in the field of genetic breeding of plants. The related report shows that the research of successfully realizing the fixation of rice heterosis is based on MiMe (Mitosis instead of Meiosis) technical system. The MiMe system can transform germ cell meiosis into mitosis, thereby producing diploid male and female gametes with no recombination, no meiosis of chromosomes, and reproduction function. It mainly involves 3 simultaneous mutations of meiosis related genes, namely, pair1 gene mutation can inhibit non sister chromatid cross exchange recombination in the process of meiosis I homologous chromosome pairing, rec8 gene mutation leads to sister chromatid separation in the later stage of meiosis I, and osd1 mutation leads to skipping of meiosis II. However, the filial generation generated by the MiMe plant selfing is tetraploid, and the chromosome multiple of the filial generation is doubled for 1 time every selfing, so that the successful realization of apomixis is difficult at present to ensure that the chromosome number of each generation is not doubled, namely haploid materials are created. At present, the approaches capable of inducing haploid formation mainly comprise the steps of ectopic expression of BBM1 genes or dandelion PAR genes, parthenogenesis induction of haploids by knocking out MTL genes, haploid induction by editing centromere specific histones cenh or haploid induction by editing DMP family genes and the like. In recent years, students at home and abroad combine haploid induction technology with MiMe system to artificially create apomictic materials to fix rice heterosis, thereby bringing dawn for breeding and applying a one-line method hybrid rice. However, the system still has the defect that the fixed frequency is low or the rice setting rate is seriously influenced, and the technical bottleneck that the haploid inductivity needs to be broken through exists. Therefore, the screening and the excavation of the high-frequency haploid induction genes are of great significance for creating the first-line hybrid rice with high fixation rate and high maturing rate. Disclosure of Invention The invention aims to solve the technical problems in the prior art and provide a method for generating rice haploids and application thereof in breeding, wherein fertilization disorder genes OsFEB, osFEB2 and OsFEB3 in rice genome are knocked out simultaneously by utilizing a gene editing technology, and the obtained three-gene homozygous knockout positive line (RHG 1, rice Haploid Induced Gene 1) is a haploid induction line and has the capability of inducing generation of haploid plants for breeding. Combining RHG1 haploid induction system with MIME system, artificially creating apomictic material to fix rice heterosis. In order to solve the technical problems, the invention provides a method for producing rice haploid, which is used for obtaining rice haploid by editing one or more of OsFEB1, osFEB2 and OsFEB3 genes in rice to lose the functions of the